WO2016103531A1 - Use of nucleic acid-polysaccharide complexes having immunopotentiating activity as anti-tumor drug - Google Patents

Use of nucleic acid-polysaccharide complexes having immunopotentiating activity as anti-tumor drug Download PDF

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WO2016103531A1
WO2016103531A1 PCT/JP2014/084772 JP2014084772W WO2016103531A1 WO 2016103531 A1 WO2016103531 A1 WO 2016103531A1 JP 2014084772 W JP2014084772 W JP 2014084772W WO 2016103531 A1 WO2016103531 A1 WO 2016103531A1
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spg
seq
cells
oligodeoxynucleotide
tumor
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PCT/JP2014/084772
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French (fr)
Japanese (ja)
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石井　健
大貴 青枝
康司 小檜山
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国立研究開発法人医薬基盤・健康・栄養研究所
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Priority to US15/539,627 priority Critical patent/US20180256630A1/en
Priority to PCT/JP2014/084772 priority patent/WO2016103531A1/en
Priority to JP2016565861A priority patent/JP6536964B2/en
Publication of WO2016103531A1 publication Critical patent/WO2016103531A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a novel cancer treatment.
  • CpG ODN CpG oligonucleotide
  • TLR9 Toll-like receptor 9
  • DCs dendritic cells
  • IFNs type I interferons
  • CTL cytotoxic T lymphocyte
  • Non-patent Document 6 There are at least four types of CpG ODN, each having a different skeletal sequence and immunostimulatory properties (Non-patent Document 6).
  • D-type (also referred to as A-type) CpG ODNs typically contain one palindromic CpG motif with a phosphodiester (PO) backbone and a phosphorothioate (PS) poly-G tail, and plasmacytoid DCs (pDCs) Is activated to produce a large amount of IFN- ⁇ , but cannot induce pDC maturation or B cell activation (Non-patent Documents 7 and 8).
  • the other three types of ODN consist of a PS skeleton.
  • K-type (also referred to as B-type) CpG ODN typically contains multiple CpG motifs of non-palindrome structure and strongly activates B cells to produce IL-6 and activates pDCs
  • IFN- ⁇ is hardly produced (Non-patent Documents 8 and 9).
  • Recently developed C-type and P-type CpG ODNs contain one and two palindromic CpG sequences, both of which activate B cells like K type and like D type
  • pDCs can be activated
  • C-type CpG ODN induces IFN- ⁇ production weaker than P-type CpG ODN (Non-patent Documents 10-12).
  • Patent Document 1 describes many excellent K-type CpG ODNs.
  • D-type and P-type CpG ODNs are Hoogsteen base pairs that form parallel four-stranded structures called G-tetrads, and Watson-Crick base pairs between cis and trans palindrome structures, Respectively, which are necessary for strong IFN- ⁇ production by pDCs (Non-patent Documents 12-14).
  • Such higher-order structures appear to be necessary for early endosome localization and TLR9-mediated signaling, but these are affected by product polymorphism and precipitation, thus preventing its clinical application (Non-patent document 15). Therefore, only K-type and C-type CpG ODN are generally available as human immunotherapeutic agents and vaccine adjuvants (Non-patent Documents 16 and 17).
  • K-type CpG ODN enhances the immunogenicity of vaccines targeting infectious diseases and cancer in human clinical trials (Non-Patent Documents 6 and 16), but for optimal adjuvant effect, antigen and K-type CpG Chemical and physical linkage between the ODN is required.
  • Schizophyllan a soluble ⁇ -1,3-glucan derived from Schizophyllum commune, is a drug approved in Japan for 30 years as an activator of radiation therapy in cervical cancer patients.
  • Patent Document 18 Lentinan (LNT), a soluble ⁇ -1,3-glucan derived from shiitake mushroom, was approved in 1985 and used in combination with fluoropyrimidines for patients with inoperable and recurrent gastric cancer. (Non-Patent Documents 19 and 20). It has been shown that ⁇ -1,3-glucan forms a complex of triple helix structure with polydeoxyadenylic acid (dA) (Non-patent Document 21).
  • dA polydeoxyadenylic acid
  • Patent Documents 2 to 4 disclose the use of a water-soluble complex of ⁇ -1,3-glucan containing schizophyllan and a nucleic acid (gene) as a gene carrier. These documents describe that the antisense action of a gene and the resistance action to a nucleolytic enzyme (nuclease) are enhanced by forming the complex.
  • Patent Document 5 by using a polysaccharide having ⁇ -1,3-linkage as a carrier (transfection agent), a CpG sequence is contained, and a phosphodiester bond is substituted with a phosphorothioate bond or a phosphorodithioate bond. It is disclosed that the action of immunostimulatory oligonucleotides is enhanced.
  • Patent Document 6 describes an immunostimulatory complex comprising an immunostimulatory oligonucleotide and ⁇ -1,3-glucan having a long ⁇ -1,6-glucoside-binding side chain. Has been.
  • Non-patent Documents 22 and 23, Patent Document 7 disclose the present inventors that mouse and humanized CpG ODN, which were conjugated with poly (dA) having a phosphodiester bond at the 5 ′ end, complexed with SPG, enhanced cytokine production and influenza vaccine. It has been shown to act as an adjuvant or a preventive or therapeutic agent for Th2 cell-related diseases.
  • poly (dA) was added to the 5 'end of each of the K-type and D-type CpG to form a complex with SPG, the activity was enhanced while maintaining the properties of the K-type and D-type, respectively.
  • Non-patent Document 24 In recent years, it has been shown that when poly (dA) having a phosphorothioate bond is linked to CpG ODN, complex formation is increased to almost 100% (Non-patent Document 24). However, no thorough testing has been done to identify the optimal humanized CpG sequence and optimize the factors to obtain the “all-in-one” activity of the four types of CpG ODN.
  • Patent Document 8 discloses a method for producing an antigen / CpG oligonucleotide / ⁇ -1,3-glucan ternary complex.
  • CpG ODN Synthetic nucleic acid CpG oligodeoxynucleotide
  • TLR9 Toll-like receptor 9
  • CpG ODN is also expected as an immunotherapeutic agent for cancer.
  • conventional CpG ODN has antitumor activity, it can only exert its effect by being administered directly to the tumor, and it has been thought that clinical application is difficult. In fact, it may be difficult to administer a drug directly to a tumor at an early stage in clinical practice. In addition, surgical treatment is required in the deep part, and the hurdle is high.
  • K3-SPG TLR9 ligand
  • PCT application PCT / JP2014 / 0748835
  • K3-SPG has been shown by experiments using mice to activate innate immunity more strongly than conventional CpG ODN without forming aggregates, and at the same time a strong adjuvant effect.
  • K3-SPG induces strong acquired immunity not only in mice but also in cynomolgus monkeys, and it was possible to overcome the difference in reactivity between mice and primates, which had been feared so far.
  • Non-Patent Document 29 Kang, X., et al.The Journal of Immunology 155,1343-1348 (1995)).
  • Cancer immunotherapy which has received particular attention, is a cancer vaccine that uses antigen-presenting cells of autologous peripheral blood that was first approved by the US Food and Drug Administration (FDA) in April 2010 for prostate cancer patients.
  • Non-patent document 30 Cancer vaccine approval couled open floodgates.Nature medicine 16,615-615 (2010);
  • Non-patent document 31 Higano, CS, et al. Cancer 115, 367079) ).
  • Non-Patent Document 32 Phan, GQ, et al. Proc. Natl. Acad. Sci. USA 100, 8372-8377 (2003);
  • Non-Patent Document 33 Camacho, L. et al. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009):
  • Non-patent document 34 Hodi. land Journal of Medicine 363,711-723 (2010)).
  • Non-patent Document 35 AZIJLI, K., et al. Anticancer Research 34, 1493- 1505 (2014);
  • Non-Patent Document 36 Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013);
  • Non-Patent Document 37 Ishida, Y., et al. The EMBO journal 11, 3877-3895. (1992);
  • Non-Patent Document 38 Topalian, SL, et al. The New England journal of medicine 366, 2443-2454 (2012)).
  • Non-Patent Document 43 Garaude, J., et al. Science translational medicine 4,120ra116 (2012);
  • Non-Patent Document 44 Martinez-Pomares. , L. et al. Trends in immunology 33, 66-70 (2012)).
  • Non-Patent Document 45 Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)).
  • K3-SPG a complex of human K-type CpG ODN K3 and beta glucan
  • K3-SPG confirmed tumor regression in tumor-bearing mice after intravenous administration, which was not effective with conventional CpG ODN (K3) (FIG. 2 (A ⁇ )).
  • K3-SPG confirmed tumor regression in tumor-bearing mice after intravenous administration, which was not effective with conventional CpG ODN (K3) (FIG. 2 (A ⁇ )).
  • the present invention has been completed, and the present inventors have further demonstrated that a peritoneal dissemination model, which is a more clinical model, also exhibits strong antitumor activity (FIG. 2g, m (FIG. 2)). 2B))
  • the present inventors did not require antigen administration for this effect, and confirmed the effect by administration of a single agent.
  • the present inventors have found that the acquired immune response is important for the antitumor effect of K3-SPG and that the type I interferon (IFN) and IL-12 induced by the innate immune response are essential. Shown using mice (FIGS. 6a, b, c (FIG. 6A)). In addition, the present inventors confirmed that CD45 negative tumor cells were accumulated in the spleen by intravenous administration of K3-SPG, and many of these cells caused cell death (necrosis or apoptosis). I made it clear.
  • mice When mice were immunized with CD45-negative cells, they exerted a strong antitumor effect, and thus it was clarified that cell death of CD45-negative cells accumulated in the spleen appears to play an important role ( 6g, h, i, j (FIG. 6B)).
  • CD8T cells activated in tumors accumulate by administering K3-SPG, and it is clear that these cells are essential for antitumor effects. It was.
  • CpG ODN that exerts an antitumor effect in systemic administration is expected to work strongly even in carcinomas that have been difficult so far. Furthermore, since CpG ODN exhibits an antitumor effect without an antigen, application as a single agent can also be expected.
  • CpG ODN has been treated as a single agent (Platesi, G., et al. Cancer research 65, 6388-6393 (2005); Manegold, C., et al. Annals ofology. Medical Oncology / ESMO 23, 72-77 (2012); Kim, YH, et al. Blood 119, 355-363 (2012); Hirsh, V., et al. Journal of clinical onology: American Society of Clini al Oncology 29, 2667-2675 (2011); Weber, JS, et al. Cancer 115, 3944-3594 (2009) or cancer vaccine adjuvant (Reed, SG, Nature medicine 19, 1597-). 1608 (2013); Perret, R., et al.
  • Clinical cancer research an official journalAmericanCertificateAmerica , 6516-6522 (2003); Lou, Y., et al., Journal of immu. other (Hagerstown, Md .: 1997) 34, 279-288 (2011); Auf, G., Clinical cancer research: an official journal of the American Assoc. , Et al. PLoS One 4, e8368 (2009); Heckelsmiller, K., et al. Journal of immunology 169, 3892-3899 (2002)).
  • K3-SPG nanoparticulate TLR9 agonist
  • SPG schizophyllan
  • K3-G B / K type CpG
  • the present inventors further examined the potential of K3-SPG single-agent immunotherapy for cancer (without using additional tumor peptides and antigens) and found that the above effects were obtained. As a result, the present invention was completed. Therefore, the present invention typically provides the following.
  • Anticancer agent alone (1) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is arranged on the 3 ′ side of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide, (B) An anticancer agent containing a complex containing ⁇ -1,3-glucan. (2) The anticancer agent according to item (1), wherein the anticancer agent is administered without a cancer antigen.
  • the reticuloendothelial system and / or lymph node includes a tumor and a phagocytic cell.
  • the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
  • the oligodeoxynucleotides are K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5)
  • the anticancer agent according to any one of items 1 to 8, selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 12 ⁇ 7).
  • the ⁇ -1,3-glucan is any one of items 1 to 9, wherein the ⁇ -1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran.
  • Anticancer drugs are selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran.
  • SPG schizophyllan
  • lentinan lentinan
  • scleroglucan scleroglucan
  • curdlan e.g., curdlan
  • parkan e.g., parkan
  • glyphoran glyphoran
  • laminaran glyphoran
  • (Reticuloendothelial system (including spleen and / or liver) and / or lymph node accumulating agent) (12) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide, (B) A composition for accumulating dead cells of cancer in the spleen, comprising a complex containing ⁇ -1,3-glucan.
  • the oligodeoxynucleotide K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5)
  • the composition according to item (12) selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  • the ⁇ -1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran, according to item (12) or (13) Composition.
  • SPG schizophyllan
  • lentinan lentinan
  • scleroglucan curdlan
  • parkan glyphoran
  • laminaran laminaran
  • composition according to any one of items (12) to (17), wherein the administration comprises systemic administration comprises systemic administration.
  • systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
  • the oligodeoxynucleotides are K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5)
  • the composition according to item (20) which is K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  • the ⁇ -1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran, according to item (20) or (21) Composition.
  • SPG schizophyllan
  • lentinan lentinan
  • scleroglucan curdlan
  • parkan glyphoran
  • laminaran laminaran
  • K3-SPG of the present invention as an antitumor agent can exert a strong antitumor effect in systemic administration that could not be overcome by conventional CpG ODN. Therefore, it is considered to be very useful from a clinical viewpoint.
  • sufficient effects innate immune response
  • Our research results show that it has a strong antitumor effect in addition to the very strong innate immune activation ability compared to CpG ODN used in clinical trials so far.
  • SPG can be expected as a useful immunotherapy drug.
  • the effect can be exerted by inducing cell death of tumor cells without requiring administration of an antigen, it can be applied to various carcinomas. From these results, K3-SPG has the potential as an innate immune activated antitumor drug that does not require an antigen.
  • FIG. 1 shows a method of complexing SPG with CpG ODN.
  • FIG. 2 (AB) shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models, including pancreatic cancer peritoneal dissemination models. Show.
  • FIG. 2A shows ai. C57BL / 6 mice were treated with EG7 cells on day 0. c.
  • FIG. 2 shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models, including pancreatic cancer peritoneal dissemination models. Show. FIG. 2B shows j to n.
  • mice (J to l) C57BL / 6 mice were inoculated with B16 cells, B16F10 cells or MC38 cells on day 0.
  • the B16 inoculated group was challenged i.e. on days 10, 12, and 14 with K3-SPG. v. Or i. t. Treated.
  • the B16F10 inoculated group was challenged i.e. on days 7, 9 and 11 with K3-SPG. v. Or i. t. Treated.
  • K3-SPG can be a cancer immunotherapy agent that does not require an antigen.
  • EG7 which is a tumor cell line
  • K3 and K3-SPG were intravenously administered three times (7th, 9th, and 11th days). Tumor size was measured from day 7 after transplantation of tumor cells.
  • FIG. 4 shows that K3-SPG targets phagocytic cells within the tumor microenvironment.
  • Ac C57BL / 6 mice were treated with EG7 on day 0.
  • mice were analyzed with an in vivo fluorescence imaging system (IVIS) and the image measured with relative fluorescence was converted to physical units of surface radiance (photons / second / cm 2 / sr).
  • IVIS in vivo fluorescence imaging system
  • White arrows indicate the tumor inoculation area (a).
  • FIG. 6A Cryosections of tumors from FIG. 6a (FIG. 6A) were stained with anti-CD3e antibody (red, EG7 staining) and Hoechst 33258 (blue, nuclear staining) and then analyzed with a fluorescence microscope (scale bar) , 100 ⁇ m).
  • White arrows indicate fluorescence positive areas.
  • FIG. 5 shows that F4 / 80 positive cells in the tumor were depleted by clodronate liposomes.
  • C57BL / 6 mice were inoculated with EG7 on day 0 and clodronate liposomes (a) or control liposomes (b) were injected i. v. And on day 7 with Alexa 647-K3-SPG i. v. Treated.
  • FIG. 6 (AB) shows that both IL-12 and IFN are important for their potential role in tumor shrinkage and their immunogenic cell death.
  • FIG. 6A shows a to f.
  • K Tumor volume at day 25 and the number of OVA 257-264 specific tetramers + CD8 T cells are represented by bar graphs and scatter paper diagrams, respectively. * P ⁇ 0.05 (t test).
  • FIG. 7 shows that IFN- ⁇ was detected in the tumor microenvironment.
  • IFN- ⁇ GFP mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-CD11b antibody, anti-CD169 antibody, anti-F4 / 80 antibody, anti-MARCO antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope. (Scale bar, 100 ⁇ m).
  • IFN- ⁇ positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P ⁇ 0.05 (t test).
  • FIG. 8 shows that IL12-p40 was detected in the tumor microenvironment.
  • A C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-IL12-p40 antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 ⁇ m).
  • B IL12-p40 positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P ⁇ 0.05 (t test).
  • FIG. 9 shows that CD45 negative cells are derived from tumor cells but not from host cells.
  • FIG. 10 shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 + DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required.
  • FIG. 10A shows a to c.
  • C57BL / 6 knockout mice (a), as well as Batf3 heterozygous and Batf3 knockout mice (b), were inoculated with EG7 cells on day 0 and i.v. K3-SPG on days 7, 9, and 11.
  • v. Treated black arrow.
  • C C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9 and 11 with K3-SPG i. d. Or i. v. Treated with.
  • FIG. 10 shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 + DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required.
  • FIG. 10B shows de.
  • FIG. 11 shows a schematic paper diagram of the experimental system.
  • WT and Il12p40-Ifnar2 DKO mice are inoculated with EG7 cells on day 0 and on days 7, 9 and 11 with K3-SPG or PBS.
  • v. Treated. On day 14, another CD8 ⁇ + T cell was purified from the spleen of these mice, labeled with Xenlight DiR®, and treated with K3-SPG (days 7, 9 and 11).
  • FIG. 12 shows the K3-SPG treatment strategy.
  • K3-SPG targeted the tumor microenvironment via the bloodstream.
  • K3-SPG targeted phagocytic cells and activated these cells.
  • IFN and IL-12 were induced by K3-SPG treatment.
  • the antigen was then released via lymph flow and blood flow. The presentation of this antigen induced a strong tumor-specific CTL.
  • the present invention provides oligodeoxynucleotides (hereinafter referred to as oligodeoxynucleotides of the present invention) containing K-type CpG oligodeoxynucleotides and polydeoxyadenylic acid (dA).
  • oligodeoxynucleotides of the present invention include those in which phosphodiester bonds are modified (for example, some or all of the phosphodiester bonds are replaced by phosphorothioate bonds).
  • the oligodeoxynucleotides of the present invention include pharmaceutically acceptable salts.
  • CpG oligonucleotide (residue) or “CpG oligodeoxynucleotide (residue)”, “CpG ODN (residue)” or simply “CpG (residue)” are used interchangeably.
  • a polynucleotide preferably an oligonucleotide, comprising at least one unmethylated CG dinucleotide sequence, which is synonymous with or without the term “residue” at the end.
  • An oligonucleotide comprising at least one CpG motif can comprise multiple CpG motifs.
  • CpG motif refers to an unmethylated dinucleotide portion of an oligonucleotide comprising a cytosine nucleotide followed by a guanosine nucleotide. 5-methylcytosine can also be used in place of cytosine.
  • polydeoxyadenylic acid and polydeoxyadenosine acid are synonymous.
  • the term “residue” means a partial structure of a compound with a higher molecular weight, but in this specification “CpG oligodeoxynucleotide (CpG ODN)” means an independent molecule or a compound with a higher molecular weight. The meaning of the partial structure is easily understood by those skilled in the art from the context. The same applies to terms relating to other partial structures contained in the oligodeoxynucleotide of the present invention such as “polydeoxyadenylic acid”.
  • CpG ODN CpG oligonucleotide
  • TLR9 Toll-like receptor 9
  • DCs dendritic cells
  • B cells B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg , AM Nature reviews.
  • Drug discovery 5,471-484 (2006) acts as an adjuvant for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) reactions ( Brazolot Millan, CL, Weera na, R., Krieg, AM, Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States3, 15A15, 15:15 (America). RS, Targoni, OS, Krieg, AM, Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997). Therefore, CpG ODN has been regarded as a potential immunotherapeutic agent for infectious diseases, cancer, asthma and hay fever (Krieg, AM Nature reviews. Drug discovery 5,471-484 (2006). Klinman, DM Nature reviews. Immunology 4, 249-258 (2004)).
  • CTL cytotoxic T lymphocyte
  • CpG ODN CpG oligodeoxynucleotide
  • K type also called B type
  • D type also called A type
  • C type and P type Advanced drug delivery reviews 61
  • K-type CpG ODNs contain multiple unmethylated CpG motifs, typically non-palindromic, and activate B cells to produce IL-6, but plasmacytoid dendritic cells (pDCs) It is a CpG ODN having structural and functional properties that hardly induce IFN- ⁇ production.
  • An unmethylated CpG motif refers to a short nucleotide sequence containing at least one cytosine (C) -guanine (G) sequence, wherein the cytosine 5-position in the cytosine-guanine sequence is not methylated.
  • CpG means unmethylated CpG unless otherwise specified.
  • the oligodeoxynucleotide of the present invention contains K-type CpG ODN, thereby activating immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) and IL-6 Activity).
  • K-type CpG ODN for example, B cells (preferably human B cells) and IL-6 Activity.
  • a number of humanized K-type CpG ODNs are known in the art (Journal of immunology 166, 2372-2377 (2001); Journal of immunology 164, 944-953 (2000); US 8,030,285B2).
  • the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. “Humanized” means having agonist activity against human TLR9.
  • the oligodeoxynucleotide of the present invention containing humanized K-type CpG ODN has an immunostimulatory activity peculiar to K-type CpG ODN (for example, an activity to activate human B cells to produce IL-6).
  • K-type CpG ODN suitably used in the present invention has a length of 10 nucleotides or more and has the formula:
  • N1, N2, N3, N4, N5 and N6 may be any nucleotide
  • the K-type CpG ODN of the present invention is 10 nucleotides or more in length and includes a nucleotide sequence of the above formula.
  • the central 4-base CpG motif (TCpGW) only needs to be contained in 10 nucleotides, and does not necessarily need to be located between N3 and N4 in the above formula.
  • N1, N2, N3, N4, N5 and N6 may be any nucleotide, and N1 and N2, N2 and N3, N3 and N4, N4 and N5, and at least one of N5 and N6
  • One (preferably one) combination may be a two-base CpG motif.
  • any two consecutive bases in the central 4 bases (4th to 7th bases) are CpG motifs,
  • the two bases can be any nucleotide.
  • the K-type CpG ODN more preferably used in the present invention contains a non-palindrome structure containing one or more CpG motifs. Further preferably used K-type CpG ODN has a non-palindrome structure containing one or more CpG motifs.
  • Humanized K-type CpG ODN is generally characterized by a 4-base CpG motif consisting of TCGA or TCGT. In many cases, two or three of these 4-base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred embodiment, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention has at least one, more preferably two or more, more preferably two or three, a 4-base CpG motif consisting of TCGA or TCGT. including. When the K-type CpG ODN has 2 or 3 4-base CpG motifs, these 4-base CpG motifs may be the same or different. However, there is no particular limitation as long as it has agonist activity against human TLR9.
  • the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention more preferably comprises the nucleotide sequence represented by SEQ ID NO: 1.
  • the length of K-type CpG ODN is particularly limited as long as the oligodeoxynucleotide of the present invention has immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6). Although not preferred, it is preferably no more than 100 nucleotides long (eg, 10-75 nucleotides long).
  • the length of K-type CpG ODN is more preferably 50 nucleotides or less (for example, 10-40 nucleotides).
  • the length of K-type CpG ODN is more preferably 30 nucleotides or less (for example, 10-25 nucleotides).
  • the length of K-type CpG ODN is most preferably 12-25 nucleotides long.
  • the length of polydeoxyadenylic acid (dA) is particularly limited as long as it is long enough to form a triple helical structure with a ⁇ -1,3-glucan (preferably lentinan or schizophyllan) chain.
  • a ⁇ -1,3-glucan preferably lentinan or schizophyllan
  • Poly dA forms a stable triple helix structure with ⁇ -1,3-glucan the longer it is, so there is theoretically no upper limit, but if it is too long, the length at the time of synthesis of oligodeoxynucleotides will vary.
  • the length of poly dA is preferably 20 to 60 nucleotides (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides long), more preferably 30 to 50 nucleotides long (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) , 42 , 43, 44, 45,
  • the oligodeoxynucleotide of the present invention has an activity of forming a triple helical structure with two schizophyllan chains by including poly dA.
  • polydeoxyadenylic acid may be described as “poly (dA)” or “poly (dA)”.
  • One molecule of the oligodeoxynucleotide of the present invention may contain a plurality of K-type CpG ODN and / or poly dA, but preferably contains one K-type CpG ODN and poly dA one by one, Most preferably, each of K-type CpG ODN and poly dA consists of one by one.
  • the oligodeoxynucleotide of the present invention is characterized in that poly dA is arranged on the 3 'side of K-type CpG ODN. With this arrangement, it is considered that the complex of the present invention (details will be described below) may also enhance the anticancer activity, but the present invention is not limited thereto. You may let them.
  • the K-type CpG ODN and poly dA may be directly linked by a covalent bond or may be linked via a spacer sequence.
  • spacer sequence is meant a nucleotide sequence comprising one or more nucleotides inserted between two adjacent components.
  • the length of the spacer sequence is such that the complex of the present invention has an immunostimulatory activity (preferably an activity to activate B cells to produce IL-6 and an activity to activate dendritic cells to produce IFN- ⁇ ).
  • an immunostimulatory activity preferably an activity to activate B cells to produce IL-6 and an activity to activate dendritic cells to produce IFN- ⁇ .
  • it is usually 1 to 10 nucleotides long, preferably 1 to 5 nucleotides long, more preferably 1 to 3 nucleotides long.
  • K-type CpG ODN and poly dA are linked by a direct covalent bond.
  • the oligodeoxynucleotide of the present invention may have an additional nucleotide sequence at its 5 'end and / or 3' end in addition to K-type CpG ODN, poly dA and an optional spacer sequence.
  • the length of the additional nucleotide sequence indicates that the complex of the present invention has an immunostimulatory activity (preferably an activity to activate B cells to produce IL-6, and a dendritic cell to produce IFN- ⁇ .
  • the length is usually 1 to 10 nucleotides, preferably 1 to 5 nucleotides, more preferably 1 to 3 nucleotides.
  • the oligodeoxynucleotide of the present invention does not contain such additional nucleotide sequences at the 5 'end and / or the 3' end. That is, the oligodeoxynucleotide of the present invention preferably comprises K-type CpG ODN, poly dA and an optional spacer sequence, and more preferably comprises K-type CpG ODN and poly dA.
  • the oligodeoxynucleotide of the present invention comprises K-type CpG ODN (specifically, for example, an oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1) and poly dA, and comprises K-type CpG ODN. Is located at the 5 ′ end of the oligodeoxynucleotide and poly dA at the 3 ′ end.
  • the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long), most preferably 30-45 nucleotides long (30, 31, 32) , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides))), which is an oligodeoxynucleotide bound to, for example, SEQ ID NO: 2 or 9 It is an oligodeoxynucleotide consisting of the nucleotide sequence represented by ⁇ 12.
  • the total length of the oligodeoxynucleotide of the present invention is generally 30 to 200 nucleotides, preferably 35 to 100 nucleotides, more preferably 40 to 80 nucleotides (specifically, 40, 41, 42, 43, 44, 45). , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides long), more preferably 50 to 70 nucleotides long (specifically, 50, 51, 52, 53, 54, 55).
  • the oligodeoxynucleotide of the present invention may be appropriately modified so as to be resistant to in vivo degradation (eg, degradation by exo or endonuclease).
  • the modification comprises a phosphorothioate modification or a phosphorodithioate modification. That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted by a phosphorothioate bond or a phosphorodithioate bond.
  • the oligodeoxynucleotide of the present invention comprises a modification of a phosphodiester bond, more preferably the modification of a phosphodiester bond is a non-phosphorothioate bond (ie, as described in WO 95/26204).
  • a modification of a phosphodiester bond is a non-phosphorothioate bond (ie, as described in WO 95/26204).
  • One of the bridging oxygen atoms is replaced by a sulfur atom). That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is replaced by a phosphorothioate bond.
  • the oligodeoxynucleotide of the present invention preferably contains a modification by phosphorothioate linkage or phosphorodithioate linkage in K-type CpG ODN, more preferably all of the phosphodiester linkage of K-type CpG ODN is Substituted with a phosphorothioate linkage.
  • the oligodeoxynucleotide of the present invention preferably contains a phosphorothioate bond or a phosphorodithioate bond in poly dA, and more preferably all of the phosphodiester bonds of poly dA are replaced with phosphorothioate bonds. Is done.
  • oligodeoxynucleotides including humanized K-type CpG oligodeoxynucleotides and polydeoxyadenylates of the invention, are replaced with phosphorothioate linkages.
  • the oligodeoxynucleotide of the present invention has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleotides (30, 30) at the 3 ′ end of a humanized K-type CpG oligodeoxynucleotide (eg, SEQ ID NO: 1).
  • the phosphorothioate bond in the oligodeoxynucleotide of the present invention, not only the resistance to degradation but also the immunostimulatory activity (for example, the activity of activating pDC to produce IFN- ⁇ ), and CpG- ⁇ -1, This is because high yield of 3-glucan complex and enhancement of anticancer activity are expected.
  • the phosphorothioate bond is synonymous with the phosphorothioate skeleton
  • the phosphate diester bond is synonymous with the phosphate skeleton.
  • the oligodeoxynucleotides of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters of the above oligodeoxynucleotides.
  • the pharmaceutically acceptable salts of the oligodeoxynucleotide of the present invention are preferably alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum Metal salts such as salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl esters Salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine Salt, piperazine salt Amine salts such as
  • the oligodeoxynucleotide of the present invention may be in any form of single strand, double strand, and triple strand, but is preferably single strand.
  • the oligodeoxynucleotide of the present invention is preferably isolated. “Isolated” means that an operation to remove factors other than the target component has been performed, and that the naturally occurring state has been removed.
  • the purity of the “isolated oligodeoxynucleotide” (percentage of the desired oligodeoxynucleotide weight in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more, more preferably 90% or more, More preferably, it is 99% or more.
  • the oligodeoxynucleotide of the present invention has excellent immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6), and thus is useful as an immunostimulator or the like. . Furthermore, since the oligodeoxynucleotide of the present invention has a property of forming a triple helix structure with two ⁇ -1,3-glucans (preferably, schizophyllan, lentinan or scleroglucan), the complex of the present invention Useful for preparation.
  • the present invention provides a complex containing the oligodeoxynucleotide of the present invention and ⁇ -1,3-glucan (hereinafter referred to as the complex of the present invention).
  • the above-mentioned oligodeoxynucleotide of the present invention contains K-type CpG ODN, it alone activates an immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) and IL -6), and immunostimulatory activity peculiar to D-type CpG ODN (for example, the activity of activating plasmacytoid dendritic cells to produce IFN- ⁇ ) is poor.
  • K-type CpG ODN for example, B cells (preferably human B cells) and IL -6
  • D-type CpG ODN for example, the activity of activating plasmacytoid dendritic cells to produce IFN- ⁇
  • ⁇ -1,3-glucan preferably lentinan, schizophyllan
  • an activation activity for example, an activity of activating plasmacytoid dendritic cells to produce IFN- ⁇
  • the complex of the present invention has an immunostimulatory activity peculiar to K-type CpG ODN (for example, an activity that activates B cells (preferably human B cells) to produce IL-6) and D-type CpG ODN.
  • Specific immunostimulatory activity for example, the activity of activating plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN- ⁇ ).
  • ⁇ -1,3-glucan used in the present invention include schizophyllan, scleroglucan, curdlan, parkan, glyphoran, lentinan, laminaran and the like.
  • the ⁇ -1,3-glucan preferably contains a large amount of 1,6-glucopyranoside branches (side chain ratio 33 to 40%), such as schizophyllan, lentinan or scleroglucan. More preferred is schizophyllan.
  • Lentinan is a known ⁇ -1,3-1,6-glucan derived from Shiitake mushroom, has a molecular formula of (C6H1005) n, and a molecular weight of about 300,000 to 700,000. It hardly dissolves in water, methanol, ethanol (95), or acetone, but dissolves in DMSO or sodium hydroxide aqueous solution, which are polar organic solvents.
  • Lentinan has an effect of enhancing activated macrophages, killer T cells, natural killer cells and antibody-dependent macrophage-mediated cytotoxicity (ADMC) activity (Hamuro, J., et al .: Immunology, 39, 551-559, 1980, Hamuro, J., et al .: Int. J. Immunopharmacol., 2, 171, 1980, Herlyn, D., et al .: Gann, 76, 37-42, 1985).
  • ADMC antibody-dependent macrophage-mediated cytotoxicity
  • Schizophyllan is a known soluble ⁇ -glucan derived from Shirohirotake.
  • SPG consists of a ⁇ - (1 ⁇ 3) -D-glucan main chain and one ⁇ - (1 ⁇ 6) -D-glucosyl side chain for each three glucoses (Tabata, K., Ito). , W., Kojima, T., Kawabata, S. and Misaki A., “Carbohydr. Res.”, 1981, 89, 1, p.
  • SPG has been used for more than 20 years as a clinical drug for intramuscular injection of immunity enhancement method against gynecological cancer (Shimizu, Chen, Kazumi, Masumi, “Biotherapy”, 1990, 4, p.
  • complex refers to a product obtained by association of a plurality of molecules through non-covalent or covalent bonds such as electrostatic bonds, van der Waals bonds, hydrogen bonds, and hydrophobic interactions. Means.
  • the complex of the present invention preferably has a triple helical structure.
  • two of the three chains forming the triple helix structure are ⁇ -1,3-glucan chains, and one is the polydeoxyadenylic acid in the oligodeoxynucleotide of the present invention. Is a chain.
  • the complex may partially include a portion that does not form a triple helical structure.
  • composition ratio of oligodeoxynucleotide and ⁇ -1,3-glucan in the complex of the present invention depends on the chain length of polydeoxyadenylic acid in the oligodeoxynucleotide, the length of ⁇ -1,3-glucan, etc. Can change. For example, when the lengths of ⁇ -1,3-glucan chains and polydeoxyadenylic acid chains are equivalent, two ⁇ -1,3-glucan chains and one oligodeoxynucleotide of the present invention Can associate to form a triple helix structure.
  • the complex of the present invention is a complex containing humanized K-type CpG ODN and ⁇ -1,3-glucan (eg, lentinan, schizophyllan, scleroglucan, curdlan, perchiman, glyphoran, laminaran), preferably Is a complex consisting of humanized K-type CpG ODN and ⁇ -1,3-glucan (eg, lentinan, schizophyllan, scleroglucan).
  • humanized K-type CpG ODN and ⁇ -1,3-glucan eg, lentinan, schizophyllan, scleroglucan
  • the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides on the 3 ′ side (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length), and oligodeoxynucleotides in which all of the phosphodiester bonds are replaced with phosphorothioate bonds, and ⁇ -1, A complex composed of 3-glucan (eg, lentinan, schizophyllan) (eg, K3-dA20-60-LNT, K3-dA20-60-SPG);
  • a complex (eg, K3-dA30-50-LNT, K3-dA30-50-SPG) composed of an oligodeoxynucleotide and ⁇ -1,3-glucan (eg, lentinan, schizophyllan), most preferably a sequence
  • the oligodeoxynucleotide consisting of the nucleotide sequence represented by No.
  • K3-dA30-45-LNT, K3-dA30-45-SPG consisting of an oligodeoxynucleotide substituted with a phosphorothioate bond and a ⁇ -1,3-glucan (eg, lentinan, schizophyllan) is there.
  • the method for preparing the complex of the present invention can be carried out in the same manner as the conditions described in Non-Patent Documents 21 to 24 and Japanese Patent Application Laid-Open No. 2008-1000091. That is, ⁇ -1,3-glucan, which originally exists as a triple helical structure in nature, is converted into an aprotic organic polar solvent (dimethyl sulfoxide (DMSO), acetonitrile, acetone, etc.) or an alkaline aqueous solution (sodium hydroxide, potassium hydroxide).
  • DMSO dimethyl sulfoxide
  • acetonitrile acetone
  • alkaline aqueous solution sodium hydroxide, potassium hydroxide
  • the solution of the single-stranded ⁇ -1,3-glucan thus obtained and the oligodeoxynucleotide solution of the present invention aqueous solution, buffer solution having a pH near neutral, or acidic buffer solution, preferably Aqueous solution or a buffered aqueous solution having a pH near neutral
  • the complex of the present invention is formed by the two ⁇ -1,3-glucan chains and the poly dA chain in the oligodeoxynucleotide forming a triple helical structure.
  • the complex By performing purification by size exclusion chromatography, ultrafiltration, dialysis and the like on the produced complex, oligodeoxynucleotides not formed in the complex can be removed. Further, by performing purification by anion exchange chromatography on the produced complex, ⁇ -1,3-glucan having no complex formed can be removed. By the above method, the complex can be appropriately purified.
  • the formation of the complex of the present invention is performed by measuring, for example, conformational change by CD (circular dichroism) spectrum, UV absorption shift by size exclusion chromatography, gel electrophoresis, microchip electrophoresis, capillary electrophoresis. Although it can confirm, it is not restricted to this.
  • CD circular dichroism
  • the mixing ratio of the oligodeoxynucleotide of the present invention and ⁇ -1,3-glucan can be appropriately set in consideration of the length of the poly dA chain and the like, but usually the molar ratio (SPG / ODN) is 0. It is 02 to 2.0, preferably 0.1 to 0.5. In a further embodiment, the molar ratio ( ⁇ -1,3-glucan (LNT, etc.) / ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25.
  • LNT is dissolved in 0.05 to 2N, preferably 0.1 to 1.5N alkaline aqueous solution (for example, 0.25N sodium hydroxide aqueous solution) and left at 1 ° C to 40 ° C for 10 hours to 4 days (for example, Left overnight at room temperature) to prepare a single-stranded LNT aqueous solution (eg, 50 mg / ml LNT aqueous solution).
  • alkaline aqueous solution for example, 0.25N sodium hydroxide aqueous solution
  • 4 days for example, Left overnight at room temperature
  • the LNT aqueous solution and a separately prepared CpG aqueous solution are mixed at a molar ratio (LNT / ODN) of 0.005 to 1.0, and then an acidic buffered aqueous solution (for example, NaH2PO4) is added to neutralize and maintained at 1-40 ° C. for 6 hours to 4 days (eg, overnight at 4 ° C.) to complete the complexation.
  • an acidic buffered aqueous solution for example, NaH2PO4
  • the composite of the present invention takes the form of bowl-like particles.
  • the particle diameter is the same as the diameter of a particle formed by ⁇ -1,3-glucan (eg, schizophyllan) used as a material naturally having a triple helical structure, and the average particle diameter is usually 10-100 nm, preferably 20-50 nm.
  • the particle diameter can be measured by a dynamic light scattering method at 80 ° C. using a Malvern Instruments Zeta Sizer after dissolving the complex in water.
  • the complex of the present invention is preferably isolated.
  • the purity of the “isolated complex” (percentage of the target complex weight in the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably Is 99% or more.
  • the complex of the present invention has excellent immunostimulatory activity in addition to anticancer activity, and in particular, immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells)) and the immunostimulatory activity peculiar to D-type CpG ODN (eg, plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) are activated to activate IFN- Since it has both of (activity which produces (alpha)), since an effect can be provided also as an immunostimulant etc., it may be advantageous.
  • immunostimulatory activity peculiar to K-type CpG ODN for example, B cells (preferably human B cells)
  • D-type CpG ODN eg, plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) are activated to activate IFN- Since it has both of (activity which produces (alpha)), since an effect can be provided also as an immunostimulant etc., it may be advantageous.
  • a K-type CpG ODN for example, a complex containing SEQ ID NO: 2, 11, 12 and SPG and a complex containing a K-type CpG ODN (for example, SEQ ID NO: 2) and SPG (K3-SPG) induces an inflammatory response.
  • Potency pan-IFN-a, IL-6, etc.
  • serum antigen-specific IgG antibody titer total IgG, IgG2c, etc.
  • IFN- antigen-specific cytokine production ability
  • the present invention provides a pharmaceutical composition comprising the oligodeoxynucleotide of the present invention or the complex of the present invention.
  • the pharmaceutical composition of the present invention can be obtained by formulating the oligodeoxynucleotide of the present invention or the complex of the present invention according to conventional means.
  • the pharmaceutical composition of the present invention comprises the oligodeoxynucleotide or complex of the present invention and a pharmacologically acceptable carrier.
  • the pharmaceutical composition may further contain an antigen.
  • Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
  • injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included.
  • Such an injection can be prepared according to a known method.
  • a method for preparing an injection it can be prepared, for example, by dissolving or suspending the oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent usually used for injection.
  • the aqueous solvent for injection include distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, and other buffer solutions.
  • the pH of such an aqueous solvent is 5 to 10, preferably 6 to 8.
  • the prepared injection solution is preferably filled in a suitable ampoule.
  • a powder formulation of the oligodeoxynucleotide or complex of the present invention can also be prepared by subjecting the suspension of the oligodeoxynucleotide or complex of the present invention to a treatment such as vacuum drying or freeze drying.
  • the oligodeoxynucleotide or complex of the present invention can be stored in a powder state, and can be used by dispersing the powder with an aqueous solvent for injection at the time of use.
  • the content of the oligodeoxynucleotide or complex of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, more preferably about 10 to 10% by weight of the whole pharmaceutical composition. About 90% by weight.
  • the pharmaceutical composition of the present invention may contain the oligodeoxynucleotide or complex of the present invention alone as an active ingredient, and contains the oligodeoxynucleotide or complex of the present invention in combination with other active ingredients. It may be.
  • the oligodeoxynucleotides and conjugates of the present invention have been found to have anticancer activity alone. Such an effect can be said to be an unexpected effect from the characteristics of the present invention that have been developed as an adjuvant. Therefore, it can be used as an adjuvant so far, that is, it is not necessary to administer with a cancer antigen, and it is not limited to a specific cancer type.
  • An anticancer agent that acts mildly on the body as an agent will be provided.
  • it since it also has immunostimulatory activity, it can also be expected to have immune activation activity against other diseases, and to have a synergistic effect on cancer patients with weak physical strength.
  • the present invention has excellent immunostimulatory activity, so the oligodeoxynucleotide, complex and pharmaceutical composition of the present invention can be used as an immunostimulator.
  • a mammal a primate such as a human, a rodent such as a mouse
  • an immune reaction in the mammal can be induced.
  • the complex of the present invention has the activity characteristics of D-type CpG ODN and stimulates peripheral blood mononuclear cells to give type I interferons (Pan-IFN- ⁇ , IFN- ⁇ 2, etc.) and type II interferons ( Since both IFN- ⁇ ) are produced in large quantities, they are useful as type I interferon production inducers, type II interferon production inducers, type I and type II interferon production inducers. Since it induces the production of both type I and type II interferons, the complex of the present invention and the pharmaceutical composition containing the same prevent or prevent diseases in which one or both of type I and type II interferons are effective. Useful for treatment.
  • Cytotoxic T lymphocytes in a subject who has received the administration can be antigen-specifically activated to prevent or treat cancer directly (as a single agent effect).
  • the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .
  • drug drug
  • drug may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity).
  • Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto.
  • a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region.
  • Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor.
  • specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
  • treatment refers to prevention of worsening of a disease or disorder when a disease or disorder (eg, cancer, allergy) occurs, preferably, maintaining the status quo. More preferably, it means reduction, further preferably elimination, which includes the ability to exert a symptom improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
  • the term “therapeutic agent (agent)” refers to any drug that can treat a target condition (for example, diseases such as cancer, allergy, etc.) in a broad sense.
  • the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
  • the pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier.
  • the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient.
  • the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution).
  • the therapeutic agent for cancer, allergy and the like includes a drug (preventive agent) used for preventing cancer, allergy and the like, or a suppressor for cancer, allergy and the like.
  • prevention means that a certain disease or disorder (for example, allergy) is prevented from becoming such a state before it becomes such a state. Diagnosis can be performed using the drug of the present invention, and for example, allergies can be prevented using the drug of the present invention, or countermeasures for prevention can be taken as necessary.
  • a certain disease or disorder for example, allergy
  • prophylactic agent refers to any agent that can prevent a target condition (for example, a disease such as allergy) in a broad sense.
  • the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, an instruction, etc.) usually divided into two or more compartments.
  • a portion to be provided eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, an instruction, etc.
  • This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use.
  • Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed).
  • the kit when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.
  • the “instruction sheet” describes the method for using the present invention for a doctor or other user.
  • This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given.
  • the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection).
  • This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received.
  • the instruction sheet is a so-called package insert and is usually provided as a paper medium, but is not limited thereto, and is in the form of, for example, an electronic medium (for example, a home page or e-mail provided on the Internet). But it can be provided.
  • the invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide.
  • an anticancer agent containing a complex comprising oligodeoxynucleotides arranged on the 3 ′ side and (b) ⁇ -1,3-glucan. In the present invention, it was found that the complex itself of the present invention acts as an anticancer agent.
  • the anticancer agent of the present invention is administered without a cancer antigen.
  • the anticancer agent of the present invention is administered so as to be delivered to the reticuloendothelial system and / or lymph nodes.
  • the reticuloendothelial system and / or lymph nodes comprise tumors and phagocytic cells.
  • the reticuloendothelial system includes the spleen and / or liver. Therefore, the anticancer agent of the present invention is characterized in that it is administered so as to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes including tumors and phagocytic cells.
  • the complexes of the invention can be delivered to tumors and phagocytic cells, where cancer dead cells can be recruited to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.
  • the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.
  • Such a delivery method may be any method, and for example, the administration may include systemic administration, but is not limited thereto.
  • Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.
  • the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), K3-dA35 (SEQ ID NO: 7), and the like.
  • ⁇ -1,3-glucan used in the present invention may be schizophyllan (SPG), lentinan, scleroglucan, curdlan, perchiman, glyforan, laminaran and the like.
  • SPG schizophyllan
  • lentinan lentinan
  • scleroglucan lentinan
  • curdlan curdlan
  • perchiman glyforan
  • laminaran laminaran
  • the complex of the present invention is K3-SPG or an analogue thereof.
  • the class itself include, but are not limited to, those having a structure similar to K3 on the CpG side and those having a structure similar to SPG on the ⁇ -glucan side.
  • the anti-cancer action is due to various mechanisms, it is not easy to conceive uses such as collecting dead cancer cells in the spleen. In particular, in systemic administration, the use for accumulating in a tumor and accumulating dead tumor cells in tissues such as the spleen is not conceivable.
  • the expression of interleukin 12 (IL12) and / or interferon (IFN) ⁇ or its promoting effect is also due to a mechanism different from the anticancer action, and interleukin 12 (IL12) and / or interferon (IFN) ) Since the expression of ⁇ or its promotion can be exerted other than anti-cancer, it is not easy to come up with each other.
  • each use of the CpG- ⁇ glucan complex of the present invention (anti-cancer use (single agent), use to accumulate cancer dead cells in the spleen, interleukin 12 (IL12) and / or interferon (IFN)) It can be said that there is a relationship that cannot be easily conceived with each other.
  • the present invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide.
  • the complex of the present invention can accumulate cancer dead cells in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes. It was.
  • treatment with a complex of the invention such as K3-SPG has been demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I.
  • CpG is targeted to phagocytic cells in the tumor microenvironment.
  • cancer dead cells Once cancer dead cells accumulate in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes, the released tumor dead cells then induce anti-tumor CTL against multiple tumor antigens.
  • the cancer cells in the body can be killed and cured as if they were attacked with a shotgun. Without wishing to be bound by theory, it is important, although not necessarily essential for K3-SPG monotherapy, to produce both IL12 and IFN-I cytokines in the tumor microenvironment.
  • the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  • the ⁇ -1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, parkan, glyphoran and laminaran.
  • the complex of the present invention is K3-SPG.
  • the reticuloendothelial system and / or lymph nodes targeted by the composition of the present invention comprises tumors and phagocytic cells.
  • the reticuloendothelial system includes the spleen and / or liver.
  • the composition of the present invention is characterized in that it is administered so as to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes containing tumors and phagocytic cells.
  • the complexes of the invention can be delivered to tumors and phagocytic cells, where cancer dead cells can be recruited to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.
  • the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.
  • Such a delivery method may be any method, and for example, the administration may include systemic administration, but is not limited thereto.
  • Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.
  • the present invention provides (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide.
  • an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide.
  • IFN interferon
  • K3-SPG monotherapy such as acting as an anti-cancer agent and other It is also important in applications.
  • examples of such treatment include, but are not limited to, cancer, chronic infectious diseases such as viruses, and prevention of viral infection.
  • the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  • the ⁇ -1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, parkan, glyphoran and laminaran.
  • the complex of the present invention is K3-SPG.
  • the present invention is provided as the above-mentioned various forms of medicaments (therapeutic or preventive).
  • the administration route of the therapeutic agent is preferably one that is effective in the treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration.
  • the administration form may be, for example, an injection, capsule, tablet, granule or the like.
  • Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers.
  • the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH.
  • the therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.
  • compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present invention comprise a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered.
  • Such carriers can be sterile liquids, such as water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals Oil, sesame oil, etc. are included.
  • Water is a preferred carrier when the drug is administered orally.
  • Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously.
  • saline solutions and aqueous dextrose and glycerol solutions are used as liquid carriers for injectable solutions.
  • Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride Sodium, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts and the like are included.
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. It is also possible to formulate the composition as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable carriers are E.I. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA).
  • compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation must be suitable for the mode of administration.
  • surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity Accelerators, flavoring agents and the like may be included.
  • the “salt” includes, for example, an anion salt formed with any acidic (eg, carboxyl) group or a cation salt formed with any basic (eg, amino) group.
  • Salts include inorganic salts or organic salts, for example, Berge et al. , J .; Pharm. Sci. , 1977, 66, 1-19. Examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and the like.
  • a “solvate” is a compound formed by a solute and a solvent. For solvates, see, for example, J. Org. Honiget al.
  • the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
  • “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.
  • various delivery systems are known, and such systems can be used to administer the therapeutic agent of the present invention to an appropriate site (eg, esophagus).
  • Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of recombinant cells capable of expressing therapeutic agents (eg, polypeptides), receptor-mediated endocytosis Use; such as the construction of therapeutic nucleic acids as part of a retroviral vector or other vector.
  • Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • an inhaler or nebulizer can be used with an aerosolizing agent and can be administered with other biologically active agents. Administration can be systemic or local.
  • the present invention can be administered by any appropriate route such as direct injection into cancer (lesion).
  • the composition can be formulated as a pharmaceutical composition adapted for human administration according to known methods. Such compositions can be administered by injection. Typically, compositions for injection administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In general, the ingredients are supplied separately or mixed together in a unit dosage form, for example in a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
  • a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product.
  • composition is to be administered by infusion
  • it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • compositions, medicament, therapeutic agent, and preventive agent of the present invention in a neutral form, salt form, or other prodrug (for example, ester).
  • pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine And those formed with free amine groups such as those derived from, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, and the like.
  • the amount of the therapeutic agent of the invention effective for the treatment of a particular disorder or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art by standard clinical techniques based on the description herein. Furthermore, in some cases, in vitro assays can be used to help identify optimal dosage ranges.
  • the exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, and within the range of any two of these values There may be.
  • the dosing interval is not particularly limited.
  • the dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient.
  • the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker is significantly decreased after administration, it may be determined that there is a therapeutic effect. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • a “patient” or “subject” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, One or more of a cat, dog, marmoset, monkey, or chimpanzee).
  • a human or non-human mammal eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, One or more of a cat, dog, marmoset, monkey, or chimpanzee.
  • the pharmaceutical composition or therapeutic agent or prophylactic agent of the present invention can be provided as a kit.
  • the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
  • a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention.
  • associated with such containers manufactured, used or sold for human administration by a government agency in a manner prescribed by the government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to indicate information indicating authorization.
  • a pharmaceutical composition comprising an ingredient of the present invention can be administered via liposomes, microparticles, or microcapsules.
  • the formulation procedure as a medicine such as a therapeutic drug and a preventive drug of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopoeia, the US Pharmacopoeia, and the pharmacopoeia of other countries. Accordingly, those skilled in the art can determine the embodiment, such as the amount to be used, without undue experimentation as described herein.
  • the oligonucleotide of the present invention can be synthesized by a standard method known in the art, for example, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). is there.
  • an automated DNA synthesizer commercially available from Biosearch, Applied Biosystems, etc.
  • it is possible to synthesize phosphorothioate oligonucleotides by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), and control pore glass polymer supports (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), etc. can also be used to prepare methylphosphonate oligonucleotides.
  • K3-dA40 SEQ ID NO: 2
  • K3-dA35 SEQ ID NO: 7
  • K3-dA30 SEQ ID NO: 6
  • K3-dA25 SEQ ID NO: 5
  • K3-dA20 SEQ ID NO: 4
  • This oligodeoxynucleotide can be obtained by using the conventional solid phase phosphoramidite method (Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. GaitOx. Synthesized.
  • Ovalbumin was purchased from Seikagaku Corporation. DQ-OVA, Alexa488-OVA, CFSE, and Lipofectamine 2000 were purchased from Invitrogen. Hoechst 33258, zymozan and curdlan were purchased from SIGMA. Zymosan-Depleted was purchased from Invivogen. Clodronate liposomes were purchased from FormuMax. Influenza split product vaccine, formalin inactivated whole virus (WIV), and purified influenza virus (H1N1) were prepared as previously described (Koyama, S., et al., Science translational medicine 2, 25ra24 (2010) ).
  • the formation of the complex was performed by using size exclusion chromatography and monitoring the shift of CpG ODN toward high molecular weight at 260 nm.
  • System Agilent 1100 series, Column: Asahipak GF7M-HQ (Shodex) 2 ligation, Flow rate: 0.8 mL / min, Buffer: 10 mM EDTA PBS, pH 7.4, Temperature: 40 ° C.).
  • EL4 and OVA expressing EL4 is a thymoma cell line of C57BL / 6J mice and was purchased from ATCC.
  • B16 (melanoma) was purchased from the Japan Collection of Research Bioresources.
  • B16F10 (melanoma) was purchased from Riken Cell Bank and MC38 (colon cancer) was Provided by Dr. JAMES Primus.
  • Pan02 pancreatic cancer was purchased from Jackson's Laboratory.
  • EL4, EG7, MC38, and Pan02 were cultured in complete RPMI (RPMI 1640 supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin, and streptomycin).
  • B16 and B16F10 were cultured in complete DMEM (DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin, and streptomycin).
  • Pan02 peritoneal seeding model In the Pan02 peritoneal seeding model, 1 ⁇ 10 6 Pan02 cells (100 ⁇ l at 1 ⁇ 10 7 cells / mL in PBS) were injected intraperitoneally. CpG treatment was started 11 days after inoculation and all tumor nodules were excised from the peritoneum of mice on day 21 and their weight (g) was then measured. The dosage for CpG treatment is as described above.
  • mice were treated with EG7 at day 0.
  • c. Inoculate and receive PBS (control), Alexa 647-K3 (30 ⁇ g), or Alexa 647-K3-SPG (10 ⁇ g) i. v. Administered.
  • PBS control
  • Alexa 647-K3 30 ⁇ g
  • Alexa 647-K3-SPG 10 ⁇ g
  • mice were analyzed with IVIS® Lumina Imaging System and analysis software (Ver. 2.6, Xenogen), and images measured with relative fluorescence were measured in physical units (photons / photon / surface radiance). Second / cm 2 / sr).
  • splenocytes were treated with K3-SPG on days 7, 9, 11 or untreated, C57BL / 6 mice carrying EG7 Or harvested on day 14 from Il12p40-Ifnar2 double knockout mice.
  • erythrocytes were lysed with ACK lysis buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA) and cells were maintained in complete RPMI.
  • CD8 ⁇ + T cells were sorted by MACS (Miltenyi Biotec). CD8 ⁇ + T cells were sorted by the negative selection method. The sorted CD8 ⁇ + T cells were then stained with Xenolight DiR®.
  • mice Stained CD8 ⁇ + T cells were injected or treated with recipient mice (EG7 on day 0, iv7 on days 0, and K3-SPG on days 7, 9, 11). C57BL / 6 mice or Il12p40-Ifnar2 double knockout mice). Twenty-four hours after transferring the stained cells, the mice were analyzed with the IVIS® Lumina Imaging System (Ver. 2.6). The region of interest was aggregated into the tumor region and the fluorescence intensity was analyzed with Living Image Software (Ver. 2.6, Xenogen).
  • C57BL / 6J mice (6-8 weeks old, female, CLEA Japan) were treated with Alexa 647-K3 (30 ⁇ g), Alexa 647-K3-SPG (10 ⁇ g), and dextran-PE (20 ⁇ g) i.e. from the tail vein.
  • Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Recovered on day 14 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained with complete RPMI. Splenocytes were isolated from H-2K b OVA tetramer (MBL), anti-CD8 ⁇ antibody (KT15), anti-TCR ⁇ antibody (H57-597), anti-CD62L antibody (MEL-14), and anti-CD44 antibody (IM7), and 7-amino.
  • MBL H-2K b OVA tetramer
  • KT15 anti-CD8 ⁇ antibody
  • KT15 anti-TCR ⁇ antibody
  • MEL-14 anti-CD44 antibody
  • IM7 anti-CD44 antibody
  • Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Harvested at day 12 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained in complete RPMI. Splenocytes were stained with anti-CD45 antibody (APC) and the number of CD45 ⁇ cells was determined by flow cytometry.
  • APC anti-CD45 antibody
  • CD45 ⁇ cells were sorted by INFLUX (BD Bioscience) from tumor bearing C57BL / 6 mice treated with K3-SPG.
  • mice C57BL / 6 mice were challenged with 5 ⁇ 10 5 CD45 ⁇ cells on day ⁇ 7. v. Administered. Seven days after immunization, mice were challenged with 5 ⁇ 10 5 EG7 cells on day 0. c. Vaccinated.
  • Mouse IL-12p40, mouse IL-13, and human IFN ⁇ levels were measured using an R & D ELISA kit.
  • Example 1 Intravenous injection of K3-SPG induces strong tumor growth inhibition without any additional tumor antigen
  • mice were inoculated with EG7 (OVA expressing mouse thymoma cell line) on the right flank on day 0, administered subcutaneously (id) near the tail base, intratumoral (it), Or three times with PBS and equimolar amounts of K3 (30 ⁇ g) or K3-SPG (10 ⁇ g) via three different routes of intravenous (iv) administration (7, 9, 11 of inoculation) days after). Tumor size was measured every 2-3 days until day 23.
  • K3-SPG forms nanoparticles with a size of about 30 nm (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)).
  • IVIS imaging uses K3-SPG instead of K3, i. v. It was revealed that it accumulated at the tumor site 1 hour after administration (FIG. 4a). It was observed that the accumulation of K3-SPG in the tumor is well related to the effectiveness of CpG monotherapy treatment for tumor shrinkage (FIG. 2).
  • IHC immunohistochemistry
  • EG7 cells express CD3e on their surface (because EG7 is derived from a thymoma cell line), but K3-SPG does not associate with CD3e, which is taken up by non-tumor cells. It showed that. Nanoparticles choose to be taken up by phagocytic cells such as macrophages and dendritic cells (DC), and these cells can be labeled with TRITC-dextran in vivo. Therefore, the inventor injected TRITC-dextran intravenously with fluorescently stained K3, K3-SPG, or SPG and tested for their coexistence by IHC (FIGS. 4d, e, f). i. v.
  • K3-SPGs do not associate with dextran and we speculate that they accumulated passively in the space within the tumor tissue via the vascular permeability and retention (EPR) effect. ing. K3-SPG i. v.
  • EPR vascular permeability and retention
  • K3-SPG i. v. To test the importance of phagocytes for treatment, we injected clodronate liposomes intravenously. The present inventors usually used 100 nm clodronate liposomes instead of 200-300 nm liposomes and injected them to deplete phagocytes in the tumor (Pante, N. et al. Molecular biology). of the cell 13,425-434 (2002); Pante, N. et al.
  • Cytokines such as IL-12 and IFN-I include C3-SPG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)).
  • ODN Korean, AM, et al. Journal of immunology 161, 428-2434 (1998); Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, KJ. , Et al. Current opinion in molecular therapeutics 6, 166-174 (2004)). Therefore, we tested whether IL-12 and IFN-I are required for tumor reduction by treatment with K3-SPG.
  • Ll7p40 and IFNAR2-deficient mice were inoculated subcutaneously with EG7 cells on day 0 and i.p. three times with PBS or K3-SPG (10 ⁇ g) as in FIG. 2 (AB). v. Treated. Thereafter, the effect of K3-SPG on tumor shrinkage was observed. (result)
  • Example 4 K3-SPG treatment induces tumor cell death in a manner dependent on both IL12p40 and IFN-I
  • K3-SPG treatment was demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I.
  • Example 5 Released tumor dead cells induce anti-tumor CTL against multiple tumor antigens.
  • CD8 T cells are important effectors in K3-SPG-mediated tumor shrinkage
  • the results of Rag2 mice showed that the tumor suppressive effect of K3-SPG is also dependent on the adaptive immune response. Therefore, we tested CD8 T cells required for K3-SPG treatment.
  • CD8 T cell depletion markedly abrogated the antitumor effect of K3-SPG (FIG. 10a (FIG. 10A)), indicating that CD8 T cells are important effector cells in this K3-SPG treatment Yes.
  • Tumor reduction by K3-SPG also depends on Batf3 (deficient in cross-presented CD8 ⁇ + DC) (FIG. 10b (FIG.
  • K3-SPG monotherapy is CD8 ⁇ + DC-mediated crossover. It also shows that presentation has been enhanced.
  • the inventors observed a clear link between tumor invasion of CD8 T cells and tumor growth.
  • CD8 T cells are expressed as K3-SPG i. v. Accumulated in the tumor area in the group i. d. There was no accumulation in the group (FIG. 10c (FIG. 10A)).
  • CD8 T cells were purified from the spleens of these mice, stained with Xenlight DiR®, and K3-SPG treated (day 7, 9, and 11) another EG7 possessed The distribution of CD8 T cells labeled with Xenolight DiR® was analyzed by IVIS on day 15 (FIG. 11).
  • CD8 T cells from donor mice bearing untreated tumors did not accumulate at the tumor site of WT recipient mice even when treated with K3-SPG (FIG. 10d (FIG. 10B), II).
  • CD8 T cells from donor mice bearing tumors treated with K3-SPG were detected at the tumor site of recipient mice (FIG. 10d (FIG. 10B), I), and K3-SPG monotherapy was We showed that we induced anti-tumor CD8 T cells that could migrate to and invade the tumor microenvironment. These in vivo activated CD8 T cells were able to enter the tumor microenvironment of DKO recipient mice (FIG. 10e (FIG. 10B)).
  • the present inventors have shown the possibility of a novel cancer immunotherapy.
  • This is a new treatment where CpG is targeted to phagocytes in the tumor microenvironment (FIG. 12).
  • CpG Through stimulation of TLR9, CpG induces an immune response by immune cells, and in particular activates macrophages and DCs (Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, K J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004)). This activation is very important for the anti-cancer immune response.
  • phagocytes phagocytosed with this novel CpG in the tumor environment produce cytokines such as IFN and IL-12. It is very important that these cytokines are induced in the tumor environment.
  • IFN ⁇ treatment directly targeting the tumor environment reactivates CTL by moving dendritic cells into the tumor and increasing antigen cross-presentation within the tumor microenvironment.
  • These cytokines cause tumor cell death.
  • the present inventors have found that this effect is exerted by activation of innate immunity. This cell death plays a very important role. This was a link between innate and adaptive immunity. Tumor cell death is released from the tumor microenvironment, thereby inducing acquired immunity.
  • This immunogenic tumor cell death induces multiple cytotoxic T lymphocytes.
  • tumor-specifically induced CTL in vivo can invade the tumor microenvironment in response to the tumor.
  • This anti-tumor immune system may use endogenous antigens to address immune editing, a barrier to cancer immunotherapy.
  • the circulation of tumor cells after K3-SPG single agent treatment can function as a biomarker with excellent treatment effect on tumors.
  • Example 6 Formulation example
  • SEQ ID NO: 2 K3-dA 40
  • SPG 15 mg
  • 0.25N NaOH 0.25N NaOH
  • a 1 mL volume of 330 mM NaH 2 PO 4 was added to the DNA solution and then the SPG solution was added to the DNA / NaH 2 PO 4 solution and maintained at 4 ° C. overnight to complete the complexation.
  • the molar ratio (M SPG / M DNA ) can be prepared by fixing at 0.27.
  • Drugs used in the formulation can be obtained from Gene Design, Invivogen, Wako and others.
  • the present invention provides a new form of anticancer agent that can be used as a single agent. Therefore, the complex of the present invention is useful in the pharmaceutical field as an anticancer agent.

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Abstract

The present invention provides an anticancer agent to be used as a single agent. More specifically, the present invention provides an anticancer agent containing complexes that contain (a) an oligodeoxynucleotide containing a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, the polydeoxyadenylic acid being located on the 3' side of the humanized K-type CpG oligodeoxynucleotide, and (b) β-1,3-glucan. The present invention is preferably characterized in that the anticancer agent is administered without a cancer antigen.

Description

免疫賦活活性を有する核酸多糖複合体の抗腫瘍薬としての応用Application of nucleic acid polysaccharide complex with immunostimulatory activity as antitumor agent
　本発明は、新規がん治療に関する。 The present invention relates to a novel cancer treatment.
　ＣｐＧオリゴヌクレオチド（ＣｐＧ　ＯＤＮ）は、免疫賦活性のＣｐＧモチーフを含有する、短い（約２０塩基対）、一本鎖の合成ＤＮＡ断片であって、Ｔｏｌｌ様受容体９（ＴＬＲ９）の強力なアゴニストであり、樹状細胞（ＤＣｓ）及びＢ細胞を活性化して、Ｉ型インターフェロン（ＩＦＮｓ）及び炎症性サイトカインを産生させ（非特許文献１、２）、細胞傷害性Ｔリンパ球（ＣＴＬ）反応を含む、Ｔｈ１型の液性及び細胞性免疫反応のアジュバントとして作用する（非特許文献３、４）。そこで、ＣｐＧ　ＯＤＮは、感染症、癌、喘息及び花粉症に対して可能性のある免疫療法剤とみなされてきた（非特許文献２、５）。 CpG oligonucleotide (CpG ODN) is a short (about 20 base pairs), single-stranded synthetic DNA fragment containing an immunostimulatory CpG motif, and is a potent agonist of Toll-like receptor 9 (TLR9) And activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Non-Patent Documents 1 and 2), and induces cytotoxic T lymphocyte (CTL) reaction. It acts as an adjuvant for the Th1 type humoral and cellular immune responses (Non-Patent Documents 3 and 4). Therefore, CpG ODN has been regarded as a potential immunotherapeutic agent for infectious diseases, cancer, asthma and hay fever (Non-Patent Documents 2 and 5).
　骨格配列及び免疫賦活特性がそれぞれ異なる、少なくとも４つの型のＣｐＧ　ＯＤＮがある（非特許文献６）。Ｄ型（Ａ型とも呼ばれる）ＣｐＧ　ＯＤＮは、典型的には、ホスホジエステル（ＰＯ）骨格及びホスホロチオエート（ＰＳ）ポリＧテイルと共に１つの回文構造のＣｐＧモチーフを含み、形質細胞様ＤＣｓ（ｐＤＣｓ）を活性化して大量のＩＦＮ−αを産生させるが、ｐＤＣ成熟化やＢ細胞活性化を誘導できない（非特許文献７、８）。他の３つの型のＯＤＮは、ＰＳ骨格からなる。Ｋ型（Ｂ型とも呼ばれる）ＣｐＧ　ＯＤＮは、典型的には、非回文構造の、複数のＣｐＧモチーフを含有し、Ｂ細胞を強力に活性化してＩＬ−６を産生させ、ｐＤＣｓを活性化して成熟化させるが、ほとんどＩＦＮ−αを産生しない（非特許文献８、９）。近年、開発されたＣ型及びＰ型のＣｐＧ　ＯＤＮはそれぞれ１つ及び２つの回文構造ＣｐＧ配列を含有しており、双方ともＫ型の様にＢ細胞を活性化させ、Ｄ型の様にｐＤＣｓを活性化させることができるが、Ｐ型ＣｐＧ　ＯＤＮと比較して、Ｃ型ＣｐＧ　ＯＤＮは、ＩＦＮ−α産生をより弱く誘導する（非特許文献１０−１２）。特許文献１に、多数の優れたＫ型ＣｐＧ　ＯＤＮが記載されている。 There are at least four types of CpG ODN, each having a different skeletal sequence and immunostimulatory properties (Non-patent Document 6). D-type (also referred to as A-type) CpG ODNs typically contain one palindromic CpG motif with a phosphodiester (PO) backbone and a phosphorothioate (PS) poly-G tail, and plasmacytoid DCs (pDCs) Is activated to produce a large amount of IFN-α, but cannot induce pDC maturation or B cell activation (Non-patent Documents 7 and 8). The other three types of ODN consist of a PS skeleton. K-type (also referred to as B-type) CpG ODN typically contains multiple CpG motifs of non-palindrome structure and strongly activates B cells to produce IL-6 and activates pDCs However, IFN-α is hardly produced (Non-patent Documents 8 and 9). Recently developed C-type and P-type CpG ODNs contain one and two palindromic CpG sequences, both of which activate B cells like K type and like D type Although pDCs can be activated, C-type CpG ODN induces IFN-α production weaker than P-type CpG ODN (Non-patent Documents 10-12). Patent Document 1 describes many excellent K-type CpG ODNs.
　Ｄ型及びＰ型ＣｐＧ　ＯＤＮは、Ｇ−ｔｅｔｒａｄｓと呼ばれる平行４本鎖構造を形成するフーグスティーン塩基対、及びシス回文構造部位とトランス回文構造部位との間のワトソン−クリック塩基対、という高次構造をそれぞれ形成することが示されており、これらはｐＤＣｓによる強力なＩＦＮ−α産生に必要である（非特許文献１２−１４）。このような高次構造は初期エンドソームへの局在化やＴＬＲ９を介する情報伝達に必要であるようだが、これらは産物の多型性及び沈殿の影響を受け、その結果その臨床応用を妨げている（非特許文献１５）。従って、Ｋ型及びＣ型ＣｐＧ　ＯＤＮのみが、ヒト用の免疫療法剤及びワクチンアジュバントとして一般的に利用可能である（非特許文献１６及び１７）。Ｋ型ＣｐＧ　ＯＤＮは、ヒト臨床試験において、感染症及び癌を標的とするワクチンの免疫原性を高めるが（非特許文献６、１６）、最適なアジュバント効果のためには、抗原とＫ型ＣｐＧ　ＯＤＮとの間の化学的及び物理的連結が必要である。これらの結果は、４つの型（Ｋ、Ｄ、Ｐ、及びＣ）のＣｐＧ　ＯＤＮには長所及び短所があることを示しており、アグリゲーションすることなく、Ｂ細胞及びｐＤＣｓの両方を活性化する「オール・イン・ワン」ＣｐＧ　ＯＤＮの開発が期待されている。 D-type and P-type CpG ODNs are Hoogsteen base pairs that form parallel four-stranded structures called G-tetrads, and Watson-Crick base pairs between cis and trans palindrome structures, Respectively, which are necessary for strong IFN-α production by pDCs (Non-patent Documents 12-14). Such higher-order structures appear to be necessary for early endosome localization and TLR9-mediated signaling, but these are affected by product polymorphism and precipitation, thus preventing its clinical application (Non-patent document 15). Therefore, only K-type and C-type CpG ODN are generally available as human immunotherapeutic agents and vaccine adjuvants (Non-patent Documents 16 and 17). K-type CpG ODN enhances the immunogenicity of vaccines targeting infectious diseases and cancer in human clinical trials (Non-Patent Documents 6 and 16), but for optimal adjuvant effect, antigen and K-type CpG Chemical and physical linkage between the ODN is required. These results indicate that the four types (K, D, P, and C) of CpG ODN have advantages and disadvantages and activate both B cells and pDCs without aggregation. Development of “All-in-One” CpG ODN is expected.
　スエヒロタケ（Ｓｃｈｉｚｏｐｈｙｌｌｕｍ　ｃｏｍｍｕｎｅ）由来の可溶性β−１，３−グルカンであるシゾフィラン（ＳＰＧ）は、子宮頸癌患者における放射線療法の賦活薬として日本においてここ３０年に亘り認可されている医薬である（非特許文献１８）。同様に、シイタケ由来の可溶性β−１，３−グルカンであるレンチナン（ＬＮＴ）は、１９８５年に承認された医薬であり、手術不能および再発胃癌患者に対しフルオロピリミジン系薬剤との併用で使用されている（非特許文献１９、２０）。β−１，３−グルカンは、ポリデオキシアデニル酸（ｄＡ）と三重螺旋構造の複合体を形成することが示されている（非特許文献２１）。 Schizophyllan (SPG), a soluble β-1,3-glucan derived from Schizophyllum commune, is a drug approved in Japan for 30 years as an activator of radiation therapy in cervical cancer patients. Patent Document 18). Similarly, Lentinan (LNT), a soluble β-1,3-glucan derived from shiitake mushroom, was approved in 1985 and used in combination with fluoropyrimidines for patients with inoperable and recurrent gastric cancer. (Non-Patent Documents 19 and 20). It has been shown that β-1,3-glucan forms a complex of triple helix structure with polydeoxyadenylic acid (dA) (Non-patent Document 21).
　特許文献２~４には、シゾフィランを含むβ−１，３−グルカンと核酸（遺伝子）との水溶性複合体の遺伝子キャリアとしての使用が開示されている。これらの文献には、該複合体を形成することにより、遺伝子のアンチセンス作用及び核酸分解酵素（ヌクレアーゼ）への耐性作用が高められることが記載されている。 Patent Documents 2 to 4 disclose the use of a water-soluble complex of β-1,3-glucan containing schizophyllan and a nucleic acid (gene) as a gene carrier. These documents describe that the antisense action of a gene and the resistance action to a nucleolytic enzyme (nuclease) are enhanced by forming the complex.
　特許文献５には、β−１，３−結合を有する多糖類をキャリアー（トランスフェクション剤）として用いることにより、ＣｐＧ配列を含み、リン酸ジエステル結合をホスホロチオエート結合又はホスホロジチオエート結合に置換した免疫刺激性オリゴヌクレオチドの作用が高められることが開示されている。 In Patent Document 5, by using a polysaccharide having β-1,3-linkage as a carrier (transfection agent), a CpG sequence is contained, and a phosphodiester bond is substituted with a phosphorothioate bond or a phosphorodithioate bond. It is disclosed that the action of immunostimulatory oligonucleotides is enhanced.
　特許文献６には、免疫刺激性オリゴヌクレオチドと、長鎖のβ−１，６−グルコシド結合側鎖を有するβ−１，３−グルカンとからなることを特徴とする免疫刺激性複合体が記載されている。 Patent Document 6 describes an immunostimulatory complex comprising an immunostimulatory oligonucleotide and β-1,3-glucan having a long β-1,6-glucoside-binding side chain. Has been.
　本発明者らは、以前、ＳＰＧと複合体化した、５’末端にリン酸ジエステル結合を有するポリ（ｄＡ）を連結させたマウス及びヒト化したＣｐＧ　ＯＤＮが、サイトカイン産生を増強し、インフルエンザワクチンアジュバントやＴｈ２細胞関連疾患の予防または治療剤として作用することを示した（非特許文献２２、２３、特許文献７）。Ｋ型及びＤ型のそれぞれのＣｐＧの５’末端にポリ（ｄＡ）を付加し、ＳＰＧと複合体を形成すると、それぞれＫ型及びＤ型の特性を維持しつつ、その活性が増強された。しかしながら、より効果的で、費用効率が高い、前臨床及び臨床開発へ向けて、ＣｐＧ−ＳＰＧ複合体の高収率を達成することが困難であった。近年、ＣｐＧ　ＯＤＮにホスホロチオエート結合を有するポリ（ｄＡ）を連結すると、複合体形成がほとんど１００％にまで上昇することが示された（非特許文献２４）。しかしながら、最適なヒト化ＣｐＧ配列を同定し、４つの型のＣｐＧ　ＯＤＮの「オール・イン・ワン」活性を得るための因子を最適化するための綿密な試験はなされていない。 The present inventors have previously reported that mouse and humanized CpG ODN, which were conjugated with poly (dA) having a phosphodiester bond at the 5 ′ end, complexed with SPG, enhanced cytokine production and influenza vaccine. It has been shown to act as an adjuvant or a preventive or therapeutic agent for Th2 cell-related diseases (Non-patent Documents 22 and 23, Patent Document 7). When poly (dA) was added to the 5 'end of each of the K-type and D-type CpG to form a complex with SPG, the activity was enhanced while maintaining the properties of the K-type and D-type, respectively. However, it has been difficult to achieve high yields of CpG-SPG conjugates for more effective and cost effective preclinical and clinical development. In recent years, it has been shown that when poly (dA) having a phosphorothioate bond is linked to CpG ODN, complex formation is increased to almost 100% (Non-patent Document 24). However, no thorough testing has been done to identify the optimal humanized CpG sequence and optimize the factors to obtain the “all-in-one” activity of the four types of CpG ODN.
　特許文献８には、抗原／ＣｐＧオリゴヌクレオチド／β−１，３−グルカン系の三元複合体の製造方法が開示されている。 Patent Document 8 discloses a method for producing an antigen / CpG oligonucleotide / β-1,3-glucan ternary complex.
　トル様受容体９　（Ｔｏｌｌ−ｌｉｋｅ　ｒｅｃｅｐｔｏｒ　９、ＴＬＲ９）のリガンドである合成核酸ＣｐＧ　オリゴデオキシヌクレオチド　（ＣｐＧ　ＯＤＮ）は強い自然免疫活性化能を有しており、ワクチンアジュバントとして期待されている。また、単剤での投与において抗腫瘍活性を有しているため、ＣｐＧ　ＯＤＮはがんに対する免疫療法剤としても期待されている。しかしながら、従来のＣｐＧ　ＯＤＮは抗腫瘍活性を有しているものの、腫瘍に直接投与する事でしか、効果を発揮する事ができず、臨床への応用は困難であると考えられていた。実際に、臨床の現場では初期の段階での腫瘍に直接薬剤を投与する事は困難であると考えられる。また、深部においては外科的処置も必要となり、そのハードルは高い。 Synthetic nucleic acid CpG oligodeoxynucleotide (CpG ODN), which is a ligand of Toll-like receptor 9, (TLR9) has strong innate immunity activation ability and is expected as a vaccine adjuvant. In addition, since CpG ODN has antitumor activity when administered as a single agent, CpG ODN is also expected as an immunotherapeutic agent for cancer. However, although conventional CpG ODN has antitumor activity, it can only exert its effect by being administered directly to the tumor, and it has been thought that clinical application is difficult. In fact, it may be difficult to administer a drug directly to a tumor at an early stage in clinical practice. In addition, surgical treatment is required in the deep part, and the hurdle is high.
　最近本発明者らはＣｐＧ　ＯＤＮを多糖のベータグルカンで包んだ、新たなＴＬＲ９のリガンド　（Ｋ３−ＳＰＧ）の開発を行った（ＰＣＴ出願（ＰＣＴ／ＪＰ２０１４／０７４８３５）））。Ｋ３−ＳＰＧは凝集塊を作る事なく、従来型のＣｐＧ　ＯＤＮに比べ、強く自然免疫を活性化する事、同時に強いアジュバント効果をマウスを用いた実験により示している。さらにＫ３−ＳＰＧは、マウスのみならずカニクイザルにおいても強い獲得免疫を誘導する事が明らかとなり、これまで懸念されていたマウスと霊長類での反応性の違いを克服できた。 Recently, the present inventors have developed a new TLR9 ligand (K3-SPG) in which CpG ODN is wrapped in a polysaccharide beta-glucan (PCT application (PCT / JP2014 / 0748835))). K3-SPG has been shown by experiments using mice to activate innate immunity more strongly than conventional CpG ODN without forming aggregates, and at the same time a strong adjuvant effect. Furthermore, it was revealed that K3-SPG induces strong acquired immunity not only in mice but also in cynomolgus monkeys, and it was possible to overcome the difference in reactivity between mice and primates, which had been feared so far.
　このように、このＣｐＧ　ＯＤＮはアジュバント剤としての用途は期待されているが、単独で医薬として使用し得るかは不明である。 Thus, although this CpG ODN is expected to be used as an adjuvant, it is unclear whether it can be used alone as a medicine.
　癌治療について言えば、１９９１年に細胞障害性Ｔ細胞が認識するがん関連抗原を同定して報告して以来（非特許文献２５＝ｖａｎ　ｄｅｒ　Ｂｒｕｇｇｅｎ　ｅｔ　ａｌ．Ｓｃｉｅｎｃｅ　（Ｎｅｗ　Ｙｏｒｋ，Ｎ．Ｙ．）２５４，１６４３−１６４７　（１９９１））、非常に多くのがん関連抗原が分子レベルで同定され、それらを標的とするがん免疫療法の臨床的適用を達成した（非特許文献２６＝Ｊａｇｅｒ，Ｅ．，ｅｔ　ａｌ．Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　ｅｘｐｅｒｉｍｅｎｔａｌ　ｍｅｄｉｃｉｎｅ　１８７，２６５−２７０　（１９９８）；非特許文献２７＝Ｊａｇｅｒ，Ｄ．ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｃｌｉｎｉｃａｌ　ｐａｔｈｏｌｏｇｙ　５４，６６９−６７４　（２００１）．；非特許文献２８＝Ｉｍａｉ，Ｋ．，ｅｔ　ａｌ．Ｂｒｉｔｉｓｈ　ｊｏｕｒｎａｌ　ｏｆ　ｃａｎｃｅｒ　１０４，３００−３０７　（２０１１）；非特許文献２９＝Ｋａｎｇ，Ｘ．，ｅｔ　ａｌ．Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｙ　１５５，１３４３−１３４８　（１９９５））。特に注目されるがん免疫療法は、２０１０年４月に前立腺がん患者に対して米国食品医薬品局（ＦＤＡ）の承認を初めて受けた、自己末梢血の抗原提示細胞を使用するがんワクチンＰｒｏｖｅｎｇｅである（非特許文献３０＝Ｃａｎｃｅｒ　ｖａｃｃｉｎｅ　ａｐｐｒｏｖａｌ　ｃｏｕｌｄ　ｏｐｅｎ　ｆｌｏｏｄｇａｔｅｓ．Ｎａｔｕｒｅ　ｍｅｄｉｃｉｎｅ　１６，６１５−６１５　（２０１０）；非特許文献３１＝Ｈｉｇａｎｏ，Ｃ．Ｓ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　１１５，３６７０−３６７９　（２００９））。その後、Ｔリンパ細胞の活性化の阻害性分子である細胞障害性Ｔリンパ球抗原４（ＣＴＬＡ−４）に対する阻害性抗体イピリムマブは、２０１１年５月に米国において、悪性黒色腫患者に対して承認された（非特許文献３２＝Ｐｈａｎ，Ｇ．Ｑ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１００，８３７２−８３７７　（２００３）；非特許文献３３＝Ｃａｍａｃｈｏ，Ｌ．Ｈ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｃｌｉｎｉｃａｌ　ｏｎｃｏｌｏｇｙ　：　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ａｍｅｒｉｃａｎ　Ｓｏｃｉｅｔｙ　ｏｆ　Ｃｌｉｎｉｃａｌ　Ｏｎｃｏｌｏｇｙ　２７，１０７５−１０８１　（２００９）；非特許文献３４＝Ｈｏｄｉ，Ｆ．Ｓ．，ｅｔ　ａｌ．Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｊｏｕｒｎａｌ　ｏｆ　Ｍｅｄｉｃｉｎｅ　３６３，７１１−７２３　（２０１０））。さらに、免疫反応阻害因子のＰＤ−１（プログラム細胞死１）受容体阻害剤であるニボルマブは、臨床試験段階である（非特許文献３５＝ＡＺＩＪＬＩ，Ｋ．，ｅｔ　ａｌ．Ａｎｔｉｃａｎｃｅｒ　Ｒｅｓｅａｒｃｈ　３４，１４９３−１５０５　（２０１４）；非特許文献３６＝Ｏｋａｚａｋｉ，Ｔ．，ｅｔ　ａｌ．Ｎａｔｕｒｅ　ｉｍｍｕｎｏｌｏｇｙ　１４，１２１２−１２１８　（２０１３）；非特許文献３７＝Ｉｓｈｉｄａ，Ｙ．，ｅｔ　ａｌ．Ｔｈｅ　ＥＭＢＯ　ｊｏｕｒｎａｌ　１１，３８８７−３８９５　（１９９２）；非特許文献３８＝Ｔｏｐａｌｉａｎ，Ｓ．Ｌ．，ｅｔ　ａｌ．Ｔｈｅ　Ｎｅｗ　Ｅｎｇｌａｎｄ　ｊｏｕｒｎａｌ　ｏｆ　ｍｅｄｉｃｉｎｅ　３６６，２４４３−２４５４　（２０１２））。 Speaking of cancer treatment, since cancer-related antigens recognized by cytotoxic T cells were identified and reported in 1991 (Non-patent Document 25 = van der Bruggen et al. Science (New York, NY). 254, 1643-1647 (1991)), a large number of cancer-related antigens have been identified at the molecular level, and clinical application of cancer immunotherapy targeting them has been achieved (Non-patent Document 26 = Jager, E., et al. The Journal of experimental medicine 187, 265-270 (1998); Non-Patent Document 27 = Jager, D. et al. Journal of clinical pathology 54, 669-674; = mai, K., et al.British journal of cancer 104,300-307 (2011); Non-Patent Document 29 = Kang, X., et al.The Journal of Immunology 155,1343-1348 (1995)). Cancer immunotherapy, which has received particular attention, is a cancer vaccine that uses antigen-presenting cells of autologous peripheral blood that was first approved by the US Food and Drug Administration (FDA) in April 2010 for prostate cancer patients. (Non-patent document 30 = Cancer vaccine approval couled open floodgates.Nature medicine 16,615-615 (2010); Non-patent document 31 = Higano, CS, et al. Cancer 115, 367079) ). Subsequently, the inhibitory antibody ipilimumab against cytotoxic T lymphocyte antigen 4 (CTLA-4), an inhibitory molecule of T lymphocyte activation, was approved in May 2011 for patients with malignant melanoma. (Non-Patent Document 32 = Phan, GQ, et al. Proc. Natl. Acad. Sci. USA 100, 8372-8377 (2003); Non-Patent Document 33 = Camacho, L. et al. H., et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 27, 1075-1081 (2009): Non-patent document 34 = Hodi. land Journal of Medicine 363,711-723 (2010)). Furthermore, nivolumab, which is a PD-1 (programmed cell death 1) receptor inhibitor of an immune response inhibitor, is in the clinical trial stage (Non-patent Document 35 = AZIJLI, K., et al. Anticancer Research 34, 1493- 1505 (2014); Non-Patent Document 36 = Okazaki, T., et al. Nature immunology 14, 1212-1218 (2013); Non-Patent Document 37 = Ishida, Y., et al. The EMBO journal 11, 3877-3895. (1992); Non-Patent Document 38 = Topalian, SL, et al. The New England journal of medicine 366, 2443-2454 (2012)).
　樹状細胞が抗がんエフェクターを効果的に導く環境の形成は、炎症部での抗がん作用を示す自然免疫のパターン分子が必要とされる（非特許文献３９＝Ｃｈｉｂａ，Ｓ．，ｅｔ　ａｌ．Ｎａｔｕｒｅ　ｉｍｍｕｎｏｌｏｇｙ　１３，８３２−８４２　（２０１２））。上記分子群およびこれに関与する過程は特定されていないが、樹状細胞は、ＣＤ４、ＣＤ８、およびＮＫなどのリンパ球を活性化する（非特許文献４０＝Ｅｎｇｅｌｈａｒｄｔ，Ｊ．Ｊ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｃｅｌｌ　２１，４０２−４１７　（２０１２））。腫瘍浸潤マクロファージ（主要関連マクロファージ：ＴＡＭ）は、炎症反応の元凶として知られている（非特許文献４１＝Ｈｕａｎｇ，Ｙ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｃｅｌｌ　１９，１−２　（２０１１））。他方で、抗がん免疫の担い手（ｐｌａｙｍａｋｅｒ）として機能する樹状細胞もまた、マクロファージと同じ骨髄細胞である（非特許文献４２＝Ｈｕａｎｇ，Ｙ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１０９，１７５６１−１７５６６　（２０１２））。これらを抗がん指向性に変える方法は見つかっていないが、腫瘍は、複雑な要因によって免疫を回避してきたと考えられている。ＴＡＭおよび樹状細胞の両方は、炎症およびパターン認識応答によって支配される（非特許文献４３＝Ｇａｒａｕｄｅ，Ｊ．，ｅｔ　ａｌ．Ｓｃｉｅｎｃｅ　ｔｒａｎｓｌａｔｉｏｎａｌ　ｍｅｄｉｃｉｎｅ　４，１２０ｒａ１１６　（２０１２）；非特許文献４４＝Ｍａｒｔｉｎｅｚ−Ｐｏｍａｒｅｓ，Ｌ．ｅｔ　ａｌ．Ｔｒｅｎｄｓ　ｉｎ　ｉｍｍｕｎｏｌｏｇｙ　３３，６６−７０　（２０１２））。免疫学的なエフェクター細胞ががん細胞と接触することは、がん細胞の攻撃のためには不可欠である（非特許文献４０＝Ｅｎｇｅｌｈａｒｄｔ，Ｊ．Ｊ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｃｅｌｌ　２１，４０２−４１７　（２０１２）；非特許文献４５＝Ｐａｌｕｃｋａ，Ｋ．ｅｔ　ａｌ．Ｎａｔｕｒｅ　ｒｅｖｉｅｗｓ．Ｃａｎｃｅｒ　１２，２６５−２７７　（２０１２））。 The formation of an environment in which dendritic cells effectively lead to anticancer effectors requires innate immunity pattern molecules that exhibit anticancer effects in the inflamed area (Non-patent Document 39 = Ciba, S., et. al. Nature immunology 13, 832-842 (2012)). Although the above-mentioned molecular group and processes involved in this are not specified, dendritic cells activate lymphocytes such as CD4, CD8, and NK (Non-patent Document 40 = Engelhardt, JJ, et al.). Cancer cell 21, 402-417 (2012)). Tumor-infiltrating macrophages (mainly related macrophages: TAM) are known as a cause of inflammatory reaction (Non-patent Document 41 = Huang, Y., et al. Cancer cell 19, 1-2 (2011)). On the other hand, dendritic cells that function as anti-cancer immunity players are also bone marrow cells that are the same as macrophages (Non-patent Document 42 = Huang, Y., et al. Proc. Natl. Acad. Sci. U.S.A. 109, 17561-17566 (2012)). Although no method has been found to turn them into anti-cancer-directed, tumors are thought to have avoided immunity due to complex factors. Both TAM and dendritic cells are governed by inflammation and pattern recognition responses (Non-Patent Document 43 = Garaude, J., et al. Science translational medicine 4,120ra116 (2012); Non-Patent Document 44 = Martinez-Pomares. , L. et al. Trends in immunology 33, 66-70 (2012)). The contact of immunological effector cells with cancer cells is indispensable for the attack of cancer cells (Non-patent Document 40 = Engelhardt, JJ, et al. Cancer cell 21, 402- 417 (2012); Non-Patent Document 45 = Palucka, K. et al. Nature reviews. Cancer 12, 265-277 (2012)).
ＵＳ　８，０３０，２８５　Ｂ２US 8,030,285 B2 ＷＯ　０１／０３４２０７　Ａ１WO 01/034207 A1 ＷＯ　０２／０７２１５２　Ａ１WO 02/0721152 A1 特開２００４−１０７２７２号公報JP 2004-107272 A ＷＯ　２００４／１００９６５　Ａ１WO 2004/100965 A1 特開２００７−７０３０７号公報JP 2007-70307 A 特開２００８−１００９１９号公報JP 2008-1000091 A 特開２０１０−１７４１０７号公報JP 2010-174107 A
　本発明者らは、鋭意研究の結果、従来アジュバントとして開発されていたＣｐＧ−βグルカン複合体（例えば、Ｋ３−ＳＰＧ（ヒト型Ｋ型ＣｐＧ　ＯＤＮであるＫ３とベータグルカンとの複合体を単剤での抗腫瘍薬として用いたところ、従来型のＣｐＧ　ＯＤＮ（Ｋ３）では効果がなかった静脈内投与において、Ｋ３−ＳＰＧは担癌状態のマウスにおける腫瘤の退縮を確認し（図２（Ａ~Ｂ））、本発明を完成させた。本発明者らは、さらに、より臨床に近いモデルである腹膜播種モデルにおいても、強力な抗腫瘍活性を示すことを実証した（図２ｇ、ｍ（図２Ｂ））。本発明者らは、この効果には抗原の投与が必要ではなく、単剤での投与において効果を確認した。 As a result of intensive studies, the present inventors have developed a CpG-β glucan complex (for example, K3-SPG (a complex of human K-type CpG ODN K3 and beta glucan) as a single agent. When used as an anti-tumor drug in the previous study, K3-SPG confirmed tumor regression in tumor-bearing mice after intravenous administration, which was not effective with conventional CpG ODN (K3) (FIG. 2 (A˜)). B)), the present invention has been completed, and the present inventors have further demonstrated that a peritoneal dissemination model, which is a more clinical model, also exhibits strong antitumor activity (FIG. 2g, m (FIG. 2)). 2B)) The present inventors did not require antigen administration for this effect, and confirmed the effect by administration of a single agent.
　さらに、本発明者らはＫ３−ＳＰＧの抗腫瘍効果には獲得免疫応答が重要である事、自然免疫応答によって誘導されるＩ型インターフェロン（ＩＦＮ）とＩＬ−１２が必須である事を遺伝子欠損マウスを用いて示した（図６ａ、ｂ、ｃ（図６Ａ））。また、本発明者らは、Ｋ３−ＳＰＧを静脈投与する事で、脾臓中にＣＤ４５陰性の腫瘍細胞が集積している事を確認し、この細胞の多くが細胞死（ｎｅｃｒｏｓｉｓまたはａｐｏｐｔｏｓｉｓ）を起こしている事を明らかにした。このＣＤ４５陰性細胞をマウスに免疫すると、強力に抗腫瘍効果を発揮したことから、脾臓に集積しているＣＤ４５陰性細胞の細胞死が重要な役割を示しているようであることを明らかにした（図６ｇ，ｈ、ｉ、ｊ（図６Ｂ））。また、本発明者らは、Ｋ３−ＳＰＧを投与する事で、腫瘍に活性化されたＣＤ８Ｔ細胞が集積する事も確認しており、これらの細胞が抗腫瘍効果には必須である事を明らかとした。 Furthermore, the present inventors have found that the acquired immune response is important for the antitumor effect of K3-SPG and that the type I interferon (IFN) and IL-12 induced by the innate immune response are essential. Shown using mice (FIGS. 6a, b, c (FIG. 6A)). In addition, the present inventors confirmed that CD45 negative tumor cells were accumulated in the spleen by intravenous administration of K3-SPG, and many of these cells caused cell death (necrosis or apoptosis). I made it clear. When mice were immunized with CD45-negative cells, they exerted a strong antitumor effect, and thus it was clarified that cell death of CD45-negative cells accumulated in the spleen appears to play an important role ( 6g, h, i, j (FIG. 6B)). In addition, the present inventors have confirmed that CD8T cells activated in tumors accumulate by administering K3-SPG, and it is clear that these cells are essential for antitumor effects. It was.
　このため、全身性投与において抗腫瘍効果を発揮するＣｐＧ　ＯＤＮの開発はこれまでに困難であった癌腫においても、強力に働くことが期待される。さらには、ＣｐＧ　ＯＤＮは抗原無しに抗腫瘍効果を発揮する事から、単剤としての応用も期待することができる。 For this reason, the development of CpG ODN that exerts an antitumor effect in systemic administration is expected to work strongly even in carcinomas that have been difficult so far. Furthermore, since CpG ODN exhibits an antitumor effect without an antigen, application as a single agent can also be expected.
　これまでに、ＣｐＧ　ＯＤＮは、単剤治療（Ｐｒａｔｅｓｉ，Ｇ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｒｅｓｅａｒｃｈ　６５，６３８８−６３９３　（２００５）；Ｍａｎｅｇｏｌｄ，Ｃ．，ｅｔ　ａｌ．Ａｎｎａｌｓ　ｏｆ　ｏｎｃｏｌｏｇｙ　：　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ｅｕｒｏｐｅａｎ　Ｓｏｃｉｅｔｙ　ｆｏｒ　Ｍｅｄｉｃａｌ　Ｏｎｃｏｌｏｇｙ　／　ＥＳＭＯ　２３，７２−７７　（２０１２）；Ｋｉｍ，Ｙ．Ｈ．，ｅｔ　ａｌ．Ｂｌｏｏｄ　１１９，３５５−３６３　（２０１２）；Ｈｉｒｓｈ，Ｖ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｃｌｉｎｉｃａｌ　ｏｎｃｏｌｏｇｙ　：　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ａｍｅｒｉｃａｎ　Ｓｏｃｉｅｔｙ　ｏｆ　Ｃｌｉｎｉｃａｌ　Ｏｎｃｏｌｏｇｙ　２９，２６６７−２６７４　（２０１１）　；Ｗｅｂｅｒ，Ｊ．Ｓ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　１１５，３９４４−３９５４　（２００９））またはがんワクチンアジュバント（Ｒｅｅｄ，Ｓ．Ｇ．，Ｎａｔｕｒｅ　ｍｅｄｉｃｉｎｅ　１９，１５９７−１６０８　（２０１３）；Ｐｅｒｒｅｔ，Ｒ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｒｅｓｅａｒｃｈ　７３，６５９７−６６０８　（２０１３）；Ｍｂｏｗ，Ｍ．Ｌ．，ｅｔ　ａｌ．Ｃｕｒｒｅｎｔ　ｏｐｉｎｉｏｎ　ｉｎ　ｉｍｍｕｎｏｌｏｇｙ　２２，４１１−４１６　（２０１０）；Ｄｕｔｈｉｅ，Ｍ．Ｓ．，ｅｔ　ａｌ．Ｉｍｍｕｎｏｌｏｇｉｃａｌ　ｒｅｖｉｅｗｓ　２３９，１７８−１９６　（２０１１））として有望な薬物であることが示されている。しかし、これまでのＣｐＧ−ＯＤＮによる抗癌剤としての治療は、腫瘍内に注射された場合にのみ、腫瘍の成長を抑制し得る（Ｓｃｈｅｔｔｉｎｉ，Ｊ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｉｍｍｕｎｏｌｏｇｙ，ｉｍｍｕｎｏｔｈｅｒａｐｙ　：　ＣＩＩ　６１，２０５５−２０６５　（２０１２）；Ｌｉｎ，Ａ．Ｙ．，ｅｔ　ａｌ．ＰＬｏＳ　Ｏｎｅ　８，ｅ６３５５０　（２０１３）；Ｉｓｈｉｉ，Ｋ．Ｊ．，ｅｔ　ａｌ．Ｃｌｉｎｉｃａｌ　ｃａｎｃｅｒ　ｒｅｓｅａｒｃｈ　：　ａｎ　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ａｍｅｒｉｃａｎ　Ａｓｓｏｃｉａｔｉｏｎ　ｆｏｒ　Ｃａｎｃｅｒ　Ｒｅｓｅａｒｃｈ　９，６５１６−６５２２　（２００３）；Ｌｏｕ，Ｙ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｔｈｅｒａｐｙ　（Ｈａｇｅｒｓｔｏｗｎ，Ｍｄ．：　１９９７）　３４，２７９−２８８　（２０１１）；Ａｕｆ，Ｇ．，Ｃｌｉｎｉｃａｌ　ｃａｎｃｅｒ　ｒｅｓｅａｒｃｈ　：　ａｎ　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ａｍｅｒｉｃａｎ　Ａｓｓｏｃｉａｔｉｏｎ　ｆｏｒ　Ｃａｎｃｅｒ　Ｒｅｓｅａｒｃｈ　７，３５４０−３５４３　（２００１）；Ｎｉｅｒｋｅｎｓ，Ｓ．，ｅｔ　ａｌ．ＰＬｏＳ　Ｏｎｅ　４，ｅ８３６８　（２００９）；Ｈｅｃｋｅｌｓｍｉｌｌｅｒ，Ｋ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｌｏｇｙ　１６９，３８９２−３８９９　（２００２））。本発明者らは、ここでＫ３−ＳＰＧというナノ粒子状のＴＬＲ９アゴニストを開発し、これはシゾフィラン（ＳＰＧ；βグルカン）およびＢ／Ｋ型ＣｐＧ（Ｋ３）複合体からなり、Ｋ３−ＳＰＧは、Ｋ３本来よりも強いワクチンアジュバント（強力なＩＦＮ−αの誘発を伴う）として機能したことを示した。本実施例において、本発明者らは、さらにがんに対するＫ３−ＳＰＧの単剤免疫療法の潜在性（さらなる腫瘍ペプチドおよび抗原を使用しない）を調べたところ、上記のような効果が得られることがわかり本発明を完成した。したがって、本発明は代表的に以下を提供する。 To date, CpG ODN has been treated as a single agent (Platesi, G., et al. Cancer research 65, 6388-6393 (2005); Manegold, C., et al. Annals ofology. Medical Oncology / ESMO 23, 72-77 (2012); Kim, YH, et al. Blood 119, 355-363 (2012); Hirsh, V., et al. Journal of clinical onology: American Society of Clini al Oncology 29, 2667-2675 (2011); Weber, JS, et al. Cancer 115, 3944-3594 (2009) or cancer vaccine adjuvant (Reed, SG, Nature medicine 19, 1597-). 1608 (2013); Perret, R., et al. Cancer research 73, 6597-6608 (2013); Mbow, ML, et al. Current opinion in immunology 22, 411-416 (2010); S., et al., Immunological reviews 239, 178-196 (2011)) is shown to be a promising drug. There. However, treatment as an anticancer agent with CpG-ODN so far can suppress tumor growth only when injected into the tumor (Schettini, J., et al. Cancer immunology, immunotherapy: CII 61, 2055). -2065 (2012); Lin, A.Y., et al. PLoS One 8, e63550 (2013); Ishii, K.J., et al. Clinical cancer research: an official journalAmericanCertificateAmerica , 6516-6522 (2003); Lou, Y., et al., Journal of immu. other (Hagerstown, Md .: 1997) 34, 279-288 (2011); Auf, G., Clinical cancer research: an official journal of the American Assoc. , Et al. PLoS One 4, e8368 (2009); Heckelsmiller, K., et al. Journal of immunology 169, 3892-3899 (2002)). The present inventors have now developed a nanoparticulate TLR9 agonist called K3-SPG, which consists of schizophyllan (SPG; β-glucan) and a B / K type CpG (K3) complex, It was shown to function as a vaccine adjuvant stronger than K3 itself (with strong induction of IFN-α). In this example, the present inventors further examined the potential of K3-SPG single-agent immunotherapy for cancer (without using additional tumor peptides and antigens) and found that the above effects were obtained. As a result, the present invention was completed. Therefore, the present invention typically provides the following.
　（抗がん剤単剤）
（１）（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
（ｂ）β—１，３−グルカンとを
含む、複合体を含む抗がん剤。
（２）前記抗がん剤は、がん抗原なしで投与されることを特徴とする、項目（１）に記載の抗がん剤。
（３）前記抗がん剤は、細網内皮系および／またはリンパ節に送達されるように投与されることを特徴とする、項目（１）または（２）に記載の抗がん剤。
（４）前記細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む、項目（３）に記載の抗がん剤。
（５）前記細網内皮系は脾臓および／または肝臓を含む、項目（３）または（４）に記載の抗がん剤。
（６）前記抗がん剤は、がん抗原なしで投与されることを特徴とする、項目（１）~（５）のいずれか１項に記載の抗がん剤。
（７）前記投与は全身性投与を含む、項目（２）~（６）のいずれか１項に記載の抗がん剤。
（８）前記全身性投与は、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与、および腫瘍内投与から選択される、項目（７）に記載の抗がん剤。
（９）前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号１２→７）からなる群より選択される、項目１~８のいずれか１項に記載の抗がん剤。
（１０）前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、項目１~９のいずれか１項に記載の抗癌剤。
（１１）前記複合体は、Ｋ３−ＳＰＧである、項目１~１０のいずれか１項に記載の抗がん剤。
（細網内皮系（脾臓および／または肝臓を含む）および／またはリンパ節集積剤）
（１２）（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
（ｂ）β—１，３−グルカンとを
含む、複合体
を含む、がんの死細胞を脾臓に集積させるための組成物。
（１３）前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）からなる群より選択される、項目（１２）に記載の組成物。
（１４）前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、項目（１２）または（１３）に記載の組成物。
（１５）前記複合体は、Ｋ３−ＳＰＧである、項目（１２）~（１４）のいずれか１項に記載の組成物。
（１６）　前記細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む、項目１２~１５のいずれか１項に記載の組成物。
（１７）　前記細網内皮系は脾臓および／または肝臓を含む、項目（１２）~（１６）のいずれか１項に記載の組成物。
（１８）前記投与は全身性投与を含む、項目（１２）~（１７）のいずれか１項に記載の組成物。
（１９）前記全身性投与は、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与、および腫瘍内投与から選択される、項目（１８）に記載の組成物。
＜インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）γの発現またはその促進のための組成物＞
（２０）（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
（ｂ）β—１，３−グルカンとを
含む、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）γの発現またはその促進のための組成物。
（２１）前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）である、項目（２０）に記載の組成物。
（２２）前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、項目（２０）または（２１）に記載の組成物。
（２３）前記複合体は、Ｋ３−ＳＰＧである、項目（２０）~（２２）のいずれか１項に記載の組成物。 (Anticancer agent alone)
(1) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is arranged on the 3 ′ side of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) An anticancer agent containing a complex containing β-1,3-glucan.
(2) The anticancer agent according to item (1), wherein the anticancer agent is administered without a cancer antigen.
(3) The anticancer agent according to item (1) or (2), wherein the anticancer agent is administered so as to be delivered to the reticuloendothelial system and / or lymph nodes.
(4) The anticancer agent according to item (3), wherein the reticuloendothelial system and / or lymph node includes a tumor and a phagocytic cell.
(5) The anticancer agent according to item (3) or (4), wherein the reticuloendothelial system includes spleen and / or liver.
(6) The anticancer agent according to any one of items (1) to (5), wherein the anticancer agent is administered without a cancer antigen.
(7) The anticancer agent according to any one of items (2) to (6), wherein the administration comprises systemic administration.
(8) The anticancer agent according to item (7), wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
(9) The oligodeoxynucleotides are K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5) The anticancer agent according to any one of items 1 to 8, selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 12 → 7).
(10) The β-1,3-glucan is any one of items 1 to 9, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran. Anticancer drugs.
(11) The anticancer agent according to any one of items 1 to 10, wherein the complex is K3-SPG.
(Reticuloendothelial system (including spleen and / or liver) and / or lymph node accumulating agent)
(12) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) A composition for accumulating dead cells of cancer in the spleen, comprising a complex containing β-1,3-glucan.
(13) The oligodeoxynucleotide K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5) The composition according to item (12), selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
(14) The β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran, according to item (12) or (13) Composition.
(15) The composition according to any one of items (12) to (14), wherein the complex is K3-SPG.
(16) The composition according to any one of items 12 to 15, wherein the reticuloendothelial system and / or lymph node includes a tumor and a phagocytic cell.
(17) The composition according to any one of items (12) to (16), wherein the reticuloendothelial system includes spleen and / or liver.
(18) The composition according to any one of items (12) to (17), wherein the administration comprises systemic administration.
(19) The composition according to item (18), wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
<Composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ>
(20) (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide An oligodeoxynucleotide,
(B) A composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, comprising β-1,3-glucan.
(21) The oligodeoxynucleotides are K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5) The composition according to item (20), which is K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
(22) The β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran, according to item (20) or (21) Composition.
(23) The composition according to any one of items (20) to (22), wherein the complex is K3-SPG.
　本発明において、上述した１または複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供され得ることが意図される。本発明のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解することにより、当業者に認識される。 In the present invention, it is intended that the above-described one or more features can be provided in combination with the combinations shown. Still further embodiments and advantages of the invention will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.
　本発明であるＫ３−ＳＰＧの抗腫瘍薬としての応用は、従来のＣｐＧ　ＯＤＮでは克服出来なかった全身性投与において強力な抗腫瘍効果を発揮する事ができる。そのため、臨床での観点からも非常に有用であると考えられる。また、ヒト細胞でも十分な効果（自然免疫応答）が確認されている事から、ヒトへの応用の可能性も高い。我々の研究結果により、これまでに臨床試験で用いられているＣｐＧ　ＯＤＮに比べ非常に強い自然免疫活性化能に加え、強い抗腫瘍効果を有している事が示されたことから、Ｋ３−ＳＰＧは有用な免疫療法薬として期待出来る。さらには、抗原の投与を必要とせず腫瘍細胞の細胞死を誘導する事で効果を発揮する事ができるため、様々な癌腫に応用出来る事が考えられる。これらの結果から、Ｋ３−ＳＰＧは抗原を必要としない自然免疫活性化型抗腫瘍薬としての可能性を有している。 The application of K3-SPG of the present invention as an antitumor agent can exert a strong antitumor effect in systemic administration that could not be overcome by conventional CpG ODN. Therefore, it is considered to be very useful from a clinical viewpoint. In addition, since sufficient effects (innate immune response) have been confirmed in human cells, there is a high possibility of application to humans. Our research results show that it has a strong antitumor effect in addition to the very strong innate immune activation ability compared to CpG ODN used in clinical trials so far. SPG can be expected as a useful immunotherapy drug. Furthermore, since the effect can be exerted by inducing cell death of tumor cells without requiring administration of an antigen, it can be applied to various carcinomas. From these results, K3-SPG has the potential as an innate immune activated antitumor drug that does not require an antigen.
図１は、ＣｐＧ　ＯＤＮとはＳＰＧの複合体化の方法を示す。FIG. 1 shows a method of complexing SPG with CpG ODN. 図２（Ａ~Ｂ）は、抗原を含まないナノ粒子状のＣｐＧ（Ｋ３−ＳＰＧ）の全身性注射が、膵臓がん腹膜播種モデルを含む多くの確立された腫瘍モデルに適用され得ることを示す。図２Ａはａ~ｉを示す。Ｃ５７ＢＬ／６マウスに、ＥＧ７細胞を０日目にｓ．ｃ．接種し、７、９および１１日目にＰＢＳ（ａ~ｃ）、Ｋ３（３０μｇ）（ｄ~ｆ）、またはＫ３−ＳＰＧ（１０μｇ）（ｇ~ｉ）を、皮内（ｉ．ｄ．）（腫瘍周囲領域）（ａ、ｄ、ｇ）、腫瘍内（ｉ．ｔ．）（ｂ、ｅ、ｈ）、または静脈内（ｉ．ｖ．）（ｃ、ｆ、ｉ）に投与した。腫瘍サイズを２３日間測定した（ｎ＝４）。各曲線は、個々のマウスを表している。矢印は、治療の時期を示している。FIG. 2 (AB) shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models, including pancreatic cancer peritoneal dissemination models. Show. FIG. 2A shows ai. C57BL / 6 mice were treated with EG7 cells on day 0. c. Inoculate and on days 7, 9 and 11 PBS (ac), K3 (30 μg) (df), or K3-SPG (10 μg) (gi) intradermal (id) (Tumor area) (a, d, g), intratumoral (it) (b, e, h), or intravenous (iv) (c, f, i). Tumor size was measured for 23 days (n = 4). Each curve represents an individual mouse. The arrow indicates the time of treatment. 図２（Ａ~Ｂ）は、抗原を含まないナノ粒子状のＣｐＧ（Ｋ３−ＳＰＧ）の全身性注射が、膵臓がん腹膜播種モデルを含む多くの確立された腫瘍モデルに適用され得ることを示す。図２Ｂはｊ~ｎを示す。（ｊ~ｌ）Ｃ５７ＢＬ／６マウスに、Ｂ１６細胞、Ｂ１６Ｆ１０細胞またはＭＣ３８細胞を０日目に接種した。Ｂ１６接種群を、Ｋ３−ＳＰＧで１０、１２および１４日目にｉ．ｖ．またはｉ．ｔ．処置した。Ｂ１６Ｆ１０接種群を、Ｋ３−ＳＰＧで７、９および１１日目にｉ．ｖ．またはｉ．ｔ．処置した。ＭＣ３８接種群を、Ｋ３−ＳＰＧで１４、１６および１８日目にｉ．ｖ．またはｉ．ｔ．処置した。エラーバーは平均＋ＳＥＭ（ｎ＝４）を表している。＊ｐ＜０．０５（ｔ検定）。（ｍ）Ｃ５７ＢＬ／６マウスに、Ｐａｎ０２細胞を０日目に腹腔内注射し、１１、１３および１５日目にＫ３またはＫ３−ＳＰＧあるいはＰＢＳ（コントロール）でｉ．ｖ．処置した。腫瘍の重量（ｇ）は２１日目を示している。＊ｐ＜０．０５（ｔ検定）。（ｎ）Ｃ５７ＢＬ／６マウスに、Ｐａｎ０２を０日目に腹腔内注射し、Ｋ３またはＫ３−ＳＰＧあるいはＰＢＳで３回ｉ．ｖ．処置した。生存率（％）を示している（ｎ＝８）。＊ｐ＜０．０５（ログランク検定）。FIG. 2 (AB) shows that systemic injection of antigen-free nanoparticulate CpG (K3-SPG) can be applied to many established tumor models, including pancreatic cancer peritoneal dissemination models. Show. FIG. 2B shows j to n. (J to l) C57BL / 6 mice were inoculated with B16 cells, B16F10 cells or MC38 cells on day 0. The B16 inoculated group was challenged i.e. on days 10, 12, and 14 with K3-SPG. v. Or i. t. Treated. The B16F10 inoculated group was challenged i.e. on days 7, 9 and 11 with K3-SPG. v. Or i. t. Treated. MC38 inoculated groups were challenged i.p. on days 14, 16 and 18 with K3-SPG. v. Or i. t. Treated. Error bars represent mean + SEM (n = 4). * P <0.05 (t test). (M) C57BL / 6 mice were injected intraperitoneally with Pan02 cells on day 0 and on days 11, 13, and 15 with K3 or K3-SPG or PBS (control). v. Treated. Tumor weight (g) indicates day 21. * P <0.05 (t test). (N) C57BL / 6 mice were injected intraperitoneally with Pan02 on day 0 and i.p. 3 times with K3 or K3-SPG or PBS. v. Treated. The survival rate (%) is shown (n = 8). * P <0.05 (log rank test). 図３は、Ｋ３−ＳＰＧの全身性投与による結果を他のコントロール、Ｋ３と比較したものである。Ｋ３−ＳＰＧの全身性投与は抗原を必要としない癌免疫療法剤となり得ることが示された。腫瘍細胞株であるＥＧ７をマウスに移植後、Ｋ３、Ｋ３−ＳＰＧを３回（７、９、１１日目）静脈内投与した。腫瘍細胞を移植後７日目から腫瘍サイズを測定した。FIG. 3 compares the results of systemic administration of K3-SPG with another control, K3. It has been shown that systemic administration of K3-SPG can be a cancer immunotherapy agent that does not require an antigen. After transplanting EG7, which is a tumor cell line, into mice, K3 and K3-SPG were intravenously administered three times (7th, 9th, and 11th days). Tumor size was measured from day 7 after transplantation of tumor cells. 図４は、Ｋ３−ＳＰＧが、腫瘍微小環境内の食細胞を標的とすることを示す。（ａ~ｃ）Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目にｓ．ｃ．接種し、ＰＢＳ（コントロール）、ＡＬＥＸＡ　６４７−Ｋ３（３０μｇ）、またはＡＬＥＸＡ　６４７−Ｋ３−ＳＰＧ（１０μｇ）を１２日目にｉ．ｖ．投与した。投与１時間後、マウスをｉｎ　ｖｉｖｏ蛍光イメージングシステム（ＩＶＩＳ）で分析し、相対蛍光で測定されたイメージを、表面放射輝度の物理単位（光子／秒／ｃｍ２／ｓｒ）に変換した。白の矢印は、腫瘍接種領域を示している（ａ）。（ｂ、ｃ）図６ａ（図６Ａ）からの腫瘍の凍結切片を、抗ＣＤ３ｅ抗体（赤、ＥＧ７染色）およびＨｏｅｃｈｓｔ　３３２５８（青、核染色）で染色し、その後蛍光顕微鏡で分析した（スケールバー、１００μｍ）。白の矢印は、蛍光陽性領域を示している。（ｄ~ｇ）Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目にｓ．ｃ．接種し、Ａｌｅｘａ　６４７−Ｋ３、Ａｌｅｘａ　６４７−Ｋ３−ＳＰＧ、またはＦＩＴＣ−ＳＰＧを、デキストラン−ＰＥと共に１２日目にｉ．ｖ．投与した。（ｄ~ｆ）注射１時間後、腫瘍の凍結切片を蛍光顕微鏡で分析した（スケールバー、１００μｍ）。（ｇ）緑、赤またはマージした細胞を計数した（３つの各腫瘍から１０視野）。エラーバーは平均＋ＳＤを表している。アスタリスクは、Ｋ３を注射されたマージ細胞数との有意差を示している。（ｈ）Ｃ５７ＢＬ／６マウス（ｎ＝３または４）に、ＥＧ７を０日目にｓ．ｃ．接種し、５日目にクロドロネートリポソームまたはコントロールリポソームをｉ．ｖ．投与した。マウスにＰＢＳ（コントロール）またはＫ３−ＳＰＧを７、９および１１日目に注射した。エラーバーは平均＋ＳＥＭを表している。矢印は治療の時期を示している。＊ｐ＜０．０５（ｔ検定）。FIG. 4 shows that K3-SPG targets phagocytic cells within the tumor microenvironment. (Ac) C57BL / 6 mice were treated with EG7 on day 0. c. Inoculate PBS (control), ALEXA 647-K3 (30 μg), or ALEXA 647-K3-SPG (10 μg) i. v. Administered. One hour after dosing, the mice were analyzed with an in vivo fluorescence imaging system (IVIS) and the image measured with relative fluorescence was converted to physical units of surface radiance (photons / second / cm 2 / sr). White arrows indicate the tumor inoculation area (a). (B, c) Cryosections of tumors from FIG. 6a (FIG. 6A) were stained with anti-CD3e antibody (red, EG7 staining) and Hoechst 33258 (blue, nuclear staining) and then analyzed with a fluorescence microscope (scale bar) , 100 μm). White arrows indicate fluorescence positive areas. (Dg) C57BL / 6 mice were treated with EG7 on day 0. c. Inoculate and received Alexa 647-K3, Alexa 647-K3-SPG, or FITC-SPG on day 12 with dextran-PE. v. Administered. (Df) One hour after injection, frozen sections of tumors were analyzed with a fluorescence microscope (scale bar, 100 μm). (G) Green, red or merged cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. The asterisk indicates a significant difference from the number of merged cells injected with K3. (H) C57BL / 6 mice (n = 3 or 4) were treated with EG7 s. c. Inoculate and on day 5 clodronate liposomes or control liposomes i. v. Administered. Mice were injected on days 7, 9 and 11 with PBS (control) or K3-SPG. Error bars represent mean + SEM. The arrow indicates the time of treatment. * P <0.05 (t test). 図５は、腫瘍におけるＦ４／８０陽性細胞がクロドロネートリポソームにより枯渇したことを示す。Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目に接種し、クロドロネートリポソーム（ａ）またはコントロールリポソーム（ｂ）を５日目にｉ．ｖ．投与し、そして、７日目にＡｌｅｘａ　６４７−Ｋ３−ＳＰＧでｉ．ｖ．処置した。処置１時間後、腫瘍の凍結切片を、抗Ｆ４／８０抗体（赤）およびＨｏｅｃｈｓｔ３３２５８（青）で染色し、その後蛍光顕微鏡で分析した（スケールバー、１００μｍ）。FIG. 5 shows that F4 / 80 positive cells in the tumor were depleted by clodronate liposomes. C57BL / 6 mice were inoculated with EG7 on day 0 and clodronate liposomes (a) or control liposomes (b) were injected i. v. And on day 7 with Alexa 647-K3-SPG i. v. Treated. One hour after treatment, frozen sections of tumors were stained with anti-F4 / 80 antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). 図６（Ａ~Ｂ）は、ＩＬ−１２およびＩＦＮの両方が、腫瘍縮小およびそれらの免疫原性細胞死における潜在的な役割に重要であることを示す。図６Ａは、ａ~ｆを示す。（ａ~ｃ）Ｉｌ１２ｐ４０ヘテロノックアウトマウス（ａ）、Ｉｆｎａｒ２ヘテロノックアウトマウス（ｂ）、およびＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウス（ｃ）に、ＥＧ７細胞を０日目にｓ．ｃ．接種し、これらのマウスを７、９および１１日目にＫ３−ＳＰＧでｉ．ｖ．処置した。エラーバーは平均＋ＳＥＭを表している（ｎ＝４）。矢印は治療の時期を示している。＊ｐ＜０．０５（ｔ検定）。（ｄ、ｆ）Ｒａｇ２ヘテロおよびノックアウトならびにＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウスに、ＥＧ７細胞を０日目に接種し、Ｋ３−ＳＰＧで３回（７、９および１１日目、黒の矢印）、６回（７、９、１１、１４、１６、１８日目、グレーの矢印）、または０回（コントロール）ｉ．ｖ．処置した。（ｅ）拡大図は４日目から２１日目を示している。FIG. 6 (AB) shows that both IL-12 and IFN are important for their potential role in tumor shrinkage and their immunogenic cell death. FIG. 6A shows a to f. (Ac) Il12p40 hetero knockout mice (a), Ifnar2 hetero knockout mice (b), and Il12p40-Ifnar2 double knockout mice (c), EG7 cells were s. c. These mice were inoculated i.v. with K3-SPG on days 7, 9 and 11. v. Treated. Error bars represent mean + SEM (n = 4). The arrow indicates the time of treatment. * P <0.05 (t test). (D, f) Rag2 hetero and knockout and Il12p40-Ifnar2 double knockout mice were inoculated with EG7 cells on day 0, 3 times with K3-SPG (7th, 9th and 11th days, black arrows), 6 times (7, 9, 11, 14, 16, 18 days, gray arrows), or 0 times (control) i. v. Treated. (E) The enlarged view shows the fourth day to the 21st day. 図６（Ａ~Ｂ）は、ＩＬ−１２およびＩＦＮの両方が、腫瘍縮小およびそれらの免疫原性細胞死における潜在的な役割に重要であることを示す。図６Ｂは、ｇ~ｋを示す。（ｇ）Ｃ５７ＢＬ／６マウスおよびＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウスに、ＥＧ７細胞を０日目にｓ．ｃ．接種し、マウスを７、９および１１日目にＫ３−ＳＰＧでｉ．ｖ．処置し、その後、１２日目に屠殺した。脾細胞を回収して抗ＣＤ４５抗体で染色し、その後、その細胞をフローサイトメトリーで分析した。（ｈ）散布図は、ＣＤ４５陰性細胞集団を示している。エラーバーは平均＋ＳＥＭを表している。＊ｐ＜０．０５（ｔ検定）。（ｉ）ＣＤ４５陰性集団を、死細胞の染色のためにＨｏｅｃｈｓｔ　３３３４２およびＰＩで染色し、その後フローサイトメトリーで分析した。棒グラフは、アポトーシス細胞、ネクローシス細胞、およびＣＤ４５陰性生細胞の集団を示している。エラーバーは平均＋ＳＤを表している（ｎ＝３）。＊ｐ＜０．０５（ｔ検定）。（ｊ）Ｃ５７ＢＬ／６マウスを、ＰＢＳまたはＣＤ４５陰性細胞で免疫化した。免疫化７日後、マウスにＥＧ７細胞を０日目にｓ．ｃ．接種し、腫瘍サイズを次の２５日間測定した（ｎ＝３）。エラーバーは平均＋ＳＥＭを表している。＊ｐ＜０．０５（ｔ検定）。（ｋ）２５日目の腫瘍体積およびＯＶＡ２５７~２６４特異的テトラマー＋ＣＤ８Ｔ細胞の数を、棒グラフおよび散布論文図によってそれぞれ表している。＊ｐ＜０．０５（ｔ検定）。FIG. 6 (AB) shows that both IL-12 and IFN are important for their potential role in tumor shrinkage and their immunogenic cell death. FIG. 6B shows g˜k. (G) C57BL / 6 mice and Il12p40-Ifnar2 double knockout mice were treated with EG7 cells on day 0. c. Inoculate and inoculate mice with K3-SPG on days 7, 9 and 11. v. Treated and then sacrificed on day 12. Splenocytes were collected and stained with anti-CD45 antibody, and then the cells were analyzed by flow cytometry. (H) Scatter plot shows CD45 negative cell population. Error bars represent mean + SEM. * P <0.05 (t test). (I) CD45 negative population was stained with Hoechst 33342 and PI for staining of dead cells and then analyzed by flow cytometry. The bar graph shows a population of apoptotic cells, necrotic cells, and live CD45 negative cells. Error bars represent mean + SD (n = 3). * P <0.05 (t test). (J) C57BL / 6 mice were immunized with PBS or CD45 negative cells. Seven days after immunization, mice were challenged with EG7 cells on day 0. c. Inoculated and tumor size was measured for the next 25 days (n = 3). Error bars represent mean + SEM. * P <0.05 (t test). (K) Tumor volume at day 25 and the number of OVA 257-264 specific tetramers + CD8 T cells are represented by bar graphs and scatter paper diagrams, respectively. * P <0.05 (t test). 図７は、ＩＦＮ−βを腫瘍微小環境において検出したことを示す。（ａ）ＩＦＮ−βＧＦＰマウスに、ＥＧ７を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧでｉ．ｄ．またはｉ．ｖ．処置した。接種１２日後、腫瘍を回収して、凍結切片を、抗ＣＤ１１ｂ抗体、抗ＣＤ１６９抗体、抗Ｆ４／８０抗体、抗ＭＡＲＣＯ抗体（赤）およびＨｏｅｃｈｓｔ　３３２５８（青）で染色し、その後蛍光顕微鏡で分析した（スケールバー、１００μｍ）。（ｂ）ＩＦＮ−β陽性細胞を計数した（３つの各腫瘍から１０視野）。エラーバーは平均＋ＳＤを表している。＊ｐ＜０．０５（ｔ検定）。FIG. 7 shows that IFN-β was detected in the tumor microenvironment. (A) IFN-βGFP mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-CD11b antibody, anti-CD169 antibody, anti-F4 / 80 antibody, anti-MARCO antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope. (Scale bar, 100 μm). (B) IFN-β positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). 図８は、ＩＬ１２−ｐ４０を腫瘍微小環境において検出したことを示す。（ａ）Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧでｉ．ｄ．またはｉ．ｖ．処置した。接種１２日後、腫瘍を回収して、凍結切片を、抗ＩＬ１２−ｐ４０抗体（赤）およびＨｏｅｃｈｓｔ　３３２５８（青）で染色し、その後蛍光顕微鏡で分析した（スケールバー、１００μｍ）。（ｂ）ＩＬ１２−ｐ４０陽性細胞を計数した（３つの各腫瘍から１０視野）。エラーバーは平均＋ＳＤを表している。＊ｐ＜０．０５（ｔ検定）。FIG. 8 shows that IL12-p40 was detected in the tumor microenvironment. (A) C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9, and 11 with K3-SPG. d. Or i. v. Treated. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-IL12-p40 antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). (B) IL12-p40 positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). 図９は、ＣＤ４５陰性細胞が腫瘍細胞に由来するが、宿主細胞に由来しないことを示す。ＧＦＰマウスにＥＧ７細胞を０日目にｓ．ｃ．接種し、そのマウスを、７、９および１１日目にＫ３−ＳＰＧでｉ．ｖ．処置し、その後１２日目に屠殺した。脾細胞を回収して、抗ＣＤ４５抗体で染色し、その後細胞をフローサイトメトリーで分析した。FIG. 9 shows that CD45 negative cells are derived from tumor cells but not from host cells. GFP mice were treated with EG7 cells on day 0. c. The mice were inoculated i.v. K3-SPG on days 7, 9 and 11. v. Treated and sacrificed on day 12 thereafter. Splenocytes were collected and stained with anti-CD45 antibody, after which the cells were analyzed by flow cytometry. 図１０（Ａ~Ｂ）は、Ｋ３−ＳＰＧ誘発性腫瘍縮小は、Ｉｌ１２、１型ＩＦＮ、Ｂａｔｆ３、ＣＤ８＋ＤＣ、および腫瘍に浸潤する強力な細胞障害性Ｔ細胞を含む自然免疫応答および適応免疫応答の両方を必要とすることを示す。図１０Ａはａ~ｃを示す。Ｃ５７ＢＬ／６ノックアウトマウス（ａ）、ならびにＢａｔｆ３ヘテロおよびＢａｔｆ３ノックアウトマウス（ｂ）に、ＥＧ７細胞を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧでｉ．ｖ．処置した（黒の矢印）。（ａ）ＣＤ８枯渇抗体（２００μｇ／マウス）を、６日目および１３日目に投与した。エラーバーは平均＋ＳＥＭを表している（ｎ＝４）。＊ｐ＜０．０５（ｔ検定）。矢印は治療の時期を示している。（ｃ）Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧをｉ．ｄ．またはｉ．ｖ．で処置した。接種１２日後、腫瘍を回収して、凍結切片を抗ＣＤ８β抗体（赤）およびＨｏｅｃｈｓｔ　３３２５８（青）で染色し、その後蛍光顕微鏡で分析した（スケールバー、１００μｍ）。ＣＤ８β陽性細胞を計数した（３つの各腫瘍から１０視野）。エラーバーは、平均＋ＳＤを表している。＊ｐ＜０．０５（ｔ検定）。FIG. 10 (AB) shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 + DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required. FIG. 10A shows a to c. C57BL / 6 knockout mice (a), as well as Batf3 heterozygous and Batf3 knockout mice (b), were inoculated with EG7 cells on day 0 and i.v. K3-SPG on days 7, 9, and 11. v. Treated (black arrow). (A) CD8 depleting antibody (200 μg / mouse) was administered on days 6 and 13. Error bars represent mean + SEM (n = 4). * P <0.05 (t test). The arrow indicates the time of treatment. (C) C57BL / 6 mice were inoculated with EG7 on day 0 and on days 7, 9 and 11 with K3-SPG i. d. Or i. v. Treated with. Twelve days after inoculation, tumors were collected and frozen sections were stained with anti-CD8β antibody (red) and Hoechst 33258 (blue) and then analyzed with a fluorescence microscope (scale bar, 100 μm). CD8β positive cells were counted (10 fields from each of 3 tumors). Error bars represent mean + SD. * P <0.05 (t test). 図１０（Ａ~Ｂ）は、Ｋ３−ＳＰＧ誘発性腫瘍縮小は、Ｉｌ１２、１型ＩＦＮ、Ｂａｔｆ３、ＣＤ８＋ＤＣ、および腫瘍に浸潤する強力な細胞障害性Ｔ細胞を含む自然免疫応答および適応免疫応答の両方を必要とすることを示す。図１０Ｂはｄ~ｅを示す。（ｄ）Ｃ５７ＢＬ／６（ＷＴ）マウスおよびＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウト（ＤＫＯ）マウスに、ＥＧ７細胞を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧでｉ．ｖ．処置した。Ｋ３−ＳＰＧまたはＰＢＳのいずれかを注射された腫瘍保有マウス由来のＣＤ８α＋Ｔ細胞を、１４日目にＸｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で染色して移し（ｉ．ｖ．）、その後、１５日目にマウスをＩＶＩＳで分析した。（Ｉ、ＩＩ）レシピエントマウス：Ｋ３−ＳＰＧでｉ．ｖ．処置されたＥＧ７保有ＷＴマウス。マウスは、Ｋ３−ＳＰＧ処置されたＣＤ８α＋Ｔ細胞が移されるか（Ｉ）、または処置されていないＣＤ８α＋Ｔ細胞を移される（ＩＩ）。（ｅ）（Ｉ、ＩＩ）レシピエントマウス：処置されていないＥＧ７保有ＷＴマウス（Ｉ）およびＫ３−ＳＰＧでｉ．ｖ．処置されたＤＫＯマウス。マウスは、Ｋ３−ＳＰＧ処置されたＣＤ８α＋Ｔ細胞を移された（Ｉ、ＩＩ）。FIG. 10 (AB) shows that K3-SPG-induced tumor shrinkage is innate and adaptive immunity including Il12, type 1 IFN, Batf3, CD8 + DC, and potent cytotoxic T cells infiltrating the tumor. Indicates that both responses are required. FIG. 10B shows de. (D) C57BL / 6 (WT) and Il12p40-Ifnar2 double knockout (DKO) mice were inoculated with EG7 cells on day 0 and i.p. K7-SPG on days 7, 9 and 11. v. Treated. CD8α + T cells from tumor-bearing mice injected with either K3-SPG or PBS were transferred on day 14 with Xenolight DiR® (iv) and then on day 15 Mice were analyzed by IVIS. (I, II) Recipient mice: i. v. Treated EG7 bearing WT mice. Mice are either transferred with K3-SPG treated CD8α + T cells (I) or untreated CD8α + T cells (II). (E) (I, II) Recipient mice: untreated EG7-bearing WT mice (I) and K3-SPG i. v. Treated DKO mice. Mice were transferred K8-SPG treated CD8α + T cells (I, II). 図１１は、実験系の模式論文図を示す。ＷＴマウスおよびＩｌ１２ｐ４０−Ｉｆｎａｒ２　ＤＫＯマウスに、ＥＧ７細胞を０日目に接種し、７、９および１１日目にＫ３−ＳＰＧまたはＰＢＳでｉ．ｖ．処置した。１４日目に、ＣＤ８α＋Ｔ細胞を、これらのマウスの脾臓から精製して、Ｘｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で標識し、そして、Ｋ３−ＳＰＧ処置（７、９および１１日目）された別のＥＧ７保有マウスに移し（接種１４日後）、その後Ｘｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で標識されたＣＤ８Ｔ細胞の分布を、１５日目にＩＶＩＳで分析した。FIG. 11 shows a schematic paper diagram of the experimental system. WT and Il12p40-Ifnar2 DKO mice are inoculated with EG7 cells on day 0 and on days 7, 9 and 11 with K3-SPG or PBS. v. Treated. On day 14, another CD8α + T cell was purified from the spleen of these mice, labeled with Xenlight DiR®, and treated with K3-SPG (days 7, 9 and 11). The distribution of CD8 T cells labeled with Xenright DiR® was analyzed by IVIS on day 15 after being transferred to EG7-bearing mice (14 days after inoculation). 図１２は、Ｋ３−ＳＰＧ処置の戦略を示す。Ｋ３−ＳＰＧは、血流を介して腫瘍微小環境を標的とした。そして、Ｋ３−ＳＰＧは食細胞を標的とし、これらの細胞を活性化した。腫瘍微小環境において、ＩＦＮおよびＩＬ−１２は、Ｋ３−ＳＰＧ処置によって誘発された。そして、抗原を、リンパ流および血流を介して放出した。この抗原の提示が強力な腫瘍特異的ＣＴＬを誘発した。FIG. 12 shows the K3-SPG treatment strategy. K3-SPG targeted the tumor microenvironment via the bloodstream. K3-SPG targeted phagocytic cells and activated these cells. In the tumor microenvironment, IFN and IL-12 were induced by K3-SPG treatment. The antigen was then released via lymph flow and blood flow. The presentation of this antigen induced a strong tumor-specific CTL.
　以下、本発明を最良の形態を示しながら説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞（例えば、英語の場合は「ａ」、「ａｎ」、「ｔｈｅ」など）は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用される全ての専門用語および科学技術用語は、本発明の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書（定義を含めて）が優先する。 Hereinafter, the present invention will be described while showing the best mode. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
　以下に本明細書において特に使用される用語の定義および／または基本的技術内容を適宜説明する。 Hereinafter, definitions of terms particularly used in this specification and / or basic technical contents will be described as appropriate.
　本発明は、Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸（ｄＡ）を含む、オリゴデオキシヌクレオチド（以下、本発明のオリゴデオキシヌクレオチドと称する。）を提供するものである。本発明のオリゴデオキシヌクレオチドにはリン酸ジエステル結合が修飾（例えば、一部又は全てのリン酸ジエステル結合がホスホロチオエート結合により置換）されているものを含む。本発明のオリゴデオキシヌクレオチドは薬学的に許容可能な塩を含む。 The present invention provides oligodeoxynucleotides (hereinafter referred to as oligodeoxynucleotides of the present invention) containing K-type CpG oligodeoxynucleotides and polydeoxyadenylic acid (dA). The oligodeoxynucleotides of the present invention include those in which phosphodiester bonds are modified (for example, some or all of the phosphodiester bonds are replaced by phosphorothioate bonds). The oligodeoxynucleotides of the present invention include pharmaceutically acceptable salts.
　本明細書において「ＣｐＧオリゴヌクレオチド（残基）」または「ＣｐＧオリゴデオキシヌクレオチド（残基）」、「ＣｐＧ　ＯＤＮ（残基）」あるいは単に「ＣｐＧ（残基）」とは、交換可能に使用され、少なくとも１つのメチル化されていないＣＧジヌクレオチド配列を含む、ポリヌクレオチド、好ましくは、オリゴヌクレオチドをいい、末尾の用語「残基」の有無に関わらず同義である。少なくとも１つのＣｐＧモチーフを含むオリゴヌクレオチドは、複数のＣｐＧモチーフを含み得る。本明細書中で使用される場合、句「ＣｐＧモチーフ」とは、シトシンヌクレオチドと、それに続くグアノシンヌクレオチドを含む、オリゴヌクレオチドのメチル化されていないジヌクレオチド部分をいう。５−メチルシトシンもまた、シトシンの代わりに使用され得る。更に、ポリデオキシアデニル酸とポリデオキシアデノシン酸（残基）は同義である。用語「残基」は、より大きな分子量の化合物の部分構造を意味するが、本明細書中、「ＣｐＧオリゴデオキシヌクレオチド（ＣｐＧ　ＯＤＮ）」が、独立した分子を意味するか、より大きな分子量の化合物の部分構造を意味するかは、当業者であれば、文脈から容易に理解可能である。「ポリデオキシアデニル酸」等、本発明のオリゴデオキシヌクレオチドに含まれる他の部分構造に関する用語についても、同様である。 In this specification, “CpG oligonucleotide (residue)” or “CpG oligodeoxynucleotide (residue)”, “CpG ODN (residue)” or simply “CpG (residue)” are used interchangeably. Refers to a polynucleotide, preferably an oligonucleotide, comprising at least one unmethylated CG dinucleotide sequence, which is synonymous with or without the term “residue” at the end. An oligonucleotide comprising at least one CpG motif can comprise multiple CpG motifs. As used herein, the phrase “CpG motif” refers to an unmethylated dinucleotide portion of an oligonucleotide comprising a cytosine nucleotide followed by a guanosine nucleotide. 5-methylcytosine can also be used in place of cytosine. Furthermore, polydeoxyadenylic acid and polydeoxyadenosine acid (residue) are synonymous. The term “residue” means a partial structure of a compound with a higher molecular weight, but in this specification “CpG oligodeoxynucleotide (CpG ODN)” means an independent molecule or a compound with a higher molecular weight. The meaning of the partial structure is easily understood by those skilled in the art from the context. The same applies to terms relating to other partial structures contained in the oligodeoxynucleotide of the present invention such as “polydeoxyadenylic acid”.
　ＣｐＧオリゴヌクレオチド（ＣｐＧ　ＯＤＮ）は、免疫賦活性のＣｐＧモチーフを含有する、短い（約２０塩基対）、一本鎖の合成ＤＮＡ断片であって、Ｔｏｌｌ様受容体９（ＴＬＲ９）の強力なアゴニストであり、樹状細胞（ＤＣｓ）およびＢ細胞を活性化して、Ｉ型インターフェロン（ＩＦＮｓ）および炎症性サイトカインを産生させ（Ｈｅｍｍｉ，Ｈ．，ｅｔ　ａｌ．Ｎａｔｕｒｅ　４０８，７４０−７４５　（２０００）；Ｋｒｉｅｇ，Ａ．Ｍ．Ｎａｔｕｒｅ　ｒｅｖｉｅｗｓ．Ｄｒｕｇ　ｄｉｓｃｏｖｅｒｙ　５，４７１−４８４　（２００６）．）、細胞傷害性Ｔリンパ球（ＣＴＬ）反応を含む、Ｔｈ１型の液性および細胞性免疫反応のアジュバントとして作用する（Ｂｒａｚｏｌｏｔ　Ｍｉｌｌａｎ，Ｃ．Ｌ．，Ｗｅｅｒａｔｎａ，Ｒ．，Ｋｒｉｅｇ，Ａ．Ｍ．，Ｓｉｅｇｒｉｓｔ，Ｃ．Ａ．＆　Ｄａｖｉｓ，Ｈ．Ｌ．Ｐｒｏｃｅｅｄｉｎｇｓ　ｏｆ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ａｃａｄｅｍｙ　ｏｆ　Ｓｃｉｅｎｃｅｓ　ｏｆ　ｔｈｅ　Ｕｎｉｔｅｄ　Ｓｔａｔｅｓ　ｏｆ　Ａｍｅｒｉｃａ　９５，１５５５３−１５５５８　（１９９８）．；Ｃｈｕ，Ｒ．Ｓ．，Ｔａｒｇｏｎｉ，Ｏ．Ｓ．，Ｋｒｉｅｇ，Ａ．Ｍ．，Ｌｅｈｍａｎｎ，Ｐ．Ｖ．＆　Ｈａｒｄｉｎｇ，Ｃ．Ｖ．Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　ｅｘｐｅｒｉｍｅｎｔａｌ　ｍｅｄｉｃｉｎｅ　１８６，１６２３−１６３１　（１９９７））。そこで、ＣｐＧ　ＯＤＮは、感染症、癌、喘息および花粉症に対して可能性のある免疫療法剤とみなされてきた（Ｋｒｉｅｇ，Ａ．Ｍ．Ｎａｔｕｒｅ　ｒｅｖｉｅｗｓ．Ｄｒｕｇ　ｄｉｓｃｏｖｅｒｙ　５，４７１−４８４　（２００６）；Ｋｌｉｎｍａｎ，Ｄ．Ｍ．Ｎａｔｕｒｅ　ｒｅｖｉｅｗｓ．Ｉｍｍｕｎｏｌｏｇｙ　４，２４９−２５８　（２００４））。 CpG oligonucleotide (CpG ODN) is a short (about 20 base pairs), single-stranded synthetic DNA fragment containing an immunostimulatory CpG motif, and is a potent agonist of Toll-like receptor 9 (TLR9) And activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg , AM Nature reviews. Drug discovery 5,471-484 (2006)), acts as an adjuvant for Th1-type humoral and cellular immune responses, including cytotoxic T lymphocyte (CTL) reactions ( Brazolot Millan, CL, Weera na, R., Krieg, AM, Siegrist, CA & Davis, HL Proceedings of the National Academy of Sciences of the United States3, 15A15, 15:15 (America). RS, Targoni, OS, Krieg, AM, Lehmann, PV & Harding, CV The Journal of experimental medicine 186, 1623-1631 (1997). Therefore, CpG ODN has been regarded as a potential immunotherapeutic agent for infectious diseases, cancer, asthma and hay fever (Krieg, AM Nature reviews. Drug discovery 5,471-484 (2006). Klinman, DM Nature reviews. Immunology 4, 249-258 (2004)).
　ＣｐＧオリゴデオキシヌクレオチド（ＣｐＧ　ＯＤＮ）は、免疫賦活性の非メチル化ＣｐＧモチーフを含有する一本鎖ＤＮＡであり、ＴＬＲ９のアゴニストである。ＣｐＧ　ＯＤＮには、骨格配列及び免疫賦活特性がそれぞれ異なる、Ｋ型（Ｂ型とも呼ばれる）、Ｄ型（Ａ型とも呼ばれる）、Ｃ型及びＰ型の４つの型がある（Ａｄｖａｎｃｅｄ　ｄｒｕｇ　ｄｅｌｉｖｅｒｙ　ｒｅｖｉｅｗｓ　６１，１９５−２０４　（２００９））。本発明のオリゴデオキシヌクレオチドは、これらのうちＫ型ＣｐＧ　ＯＤＮを含む。 CpG oligodeoxynucleotide (CpG ODN) is a single-stranded DNA containing an immunostimulatory unmethylated CpG motif and is an agonist of TLR9. There are four types of CpG ODN, K type (also called B type), D type (also called A type), C type and P type (Advanced drug delivery reviews 61), which have different skeletal sequences and immunostimulatory properties. , 195-204 (2009)). Among these, the oligodeoxynucleotide of the present invention includes K-type CpG ODN.
　Ｋ型ＣｐＧ　ＯＤＮは、典型的には非回文構造の、複数の非メチル化ＣｐＧモチーフを含有し、Ｂ細胞を活性化してＩＬ−６を産生させるが、形質細胞様樹状細胞（ｐＤＣｓ）のＩＦＮ−α産生をほとんど誘導しないという構造的及び機能的特性を有するＣｐＧ　ＯＤＮである。非メチル化ＣｐＧモチーフとは少なくとも１つのシトシン（Ｃ）−グアニン（Ｇ）配列を含む短いヌクレオチド配列であって、該シトシン−グアニン配列におけるシトシンの５位がメチル化されていないものを差す。なお、以下の説明において、ＣｐＧとは、特にことわらなり限り非メチル化ＣｐＧを意味する。従って、本発明のオリゴデオキシヌクレオチドは、Ｋ型ＣｐＧ　ＯＤＮを含むことにより、Ｋ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）を有する。当該技術分野において多数のヒト化Ｋ型ＣｐＧ　ＯＤＮが知られている（Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｌｏｇｙ　１６６，２３７２−２３７７　（２００１）；Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｌｏｇｙ　１６４，９４４−９５３　（２０００）；ＵＳ　８，０３０，２８５Ｂ２）。
　本発明のオリゴデオキシヌクレオチドに含まれるＫ型ＣｐＧ　ＯＤＮは、好ましくはヒト化されている。「ヒト化」とは、ヒトＴＬＲ９に対するアゴニスト活性を有することを意味する。従って、ヒト化Ｋ型ＣｐＧ　ＯＤＮを含む本発明のオリゴデオキシヌクレオチドは、ヒトに対してＫ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、ヒトＢ細胞を活性化してＩＬ−６を産生させる活性）を有する。本発明において好適に用いられるＫ型ＣｐＧ　ＯＤＮは、１０ヌクレオチド以上の長さであり、且つ式： K-type CpG ODNs contain multiple unmethylated CpG motifs, typically non-palindromic, and activate B cells to produce IL-6, but plasmacytoid dendritic cells (pDCs) It is a CpG ODN having structural and functional properties that hardly induce IFN-α production. An unmethylated CpG motif refers to a short nucleotide sequence containing at least one cytosine (C) -guanine (G) sequence, wherein the cytosine 5-position in the cytosine-guanine sequence is not methylated. In the following description, CpG means unmethylated CpG unless otherwise specified. Therefore, the oligodeoxynucleotide of the present invention contains K-type CpG ODN, thereby activating immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) and IL-6 Activity). A number of humanized K-type CpG ODNs are known in the art (Journal of immunology 166, 2372-2377 (2001); Journal of immunology 164, 944-953 (2000); US 8,030,285B2).
The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention is preferably humanized. “Humanized” means having agonist activity against human TLR9. Therefore, the oligodeoxynucleotide of the present invention containing humanized K-type CpG ODN has an immunostimulatory activity peculiar to K-type CpG ODN (for example, an activity to activate human B cells to produce IL-6). Have The K-type CpG ODN suitably used in the present invention has a length of 10 nucleotides or more and has the formula:
Figure JPOXMLDOC01-appb-I000001
（式中、中央のＣｐＧモチーフはメチル化されておらず、ＷはＡ又はＴであり、Ｎ１、Ｎ２、Ｎ３、Ｎ４、Ｎ５及びＮ６はいかなるヌクレオチドであってもよい）で表されるヌクレオチド配列を含む。
Figure JPOXMLDOC01-appb-I000001
(Wherein the central CpG motif is not methylated, W is A or T, and N1, N2, N3, N4, N5 and N6 may be any nucleotide) including.
　１つの実施形態において、本発明のＫ型ＣｐＧ　ＯＤＮは１０ヌクレオチド以上の長さであり、上記式のヌクレオチド配列を含む。但し、上記式中、中央の４塩基のＣｐＧモチーフ（ＴＣｐＧＷ）は１０ヌクレオチド中に含まれていれば良く、必ずしも上記式中、Ｎ３及びＮ４の間に位置する必要はない。また、上記式中、Ｎ１、Ｎ２、Ｎ３、Ｎ４、Ｎ５及びＮ６はいかなるヌクレオチドであっても良く、Ｎ１及びＮ２、Ｎ２及びＮ３、Ｎ３及びＮ４、Ｎ４及びＮ５、並びにＮ５及びＮ６の少なくともいずれか一つ（好ましくは一つ）の組み合わせは２塩基のＣｐＧモチーフであっても良い。前記４塩基のＣｐＧモチーフがＮ３及びＮ４の間に位置しない場合、上記式中、中央の４塩基（４~７番目の塩基）中の連続するいずれかの２塩基がＣｐＧモチーフであり、他の２つの塩基はいかなるヌクレオチドであっても良い。 In one embodiment, the K-type CpG ODN of the present invention is 10 nucleotides or more in length and includes a nucleotide sequence of the above formula. However, in the above formula, the central 4-base CpG motif (TCpGW) only needs to be contained in 10 nucleotides, and does not necessarily need to be located between N3 and N4 in the above formula. In the above formula, N1, N2, N3, N4, N5 and N6 may be any nucleotide, and N1 and N2, N2 and N3, N3 and N4, N4 and N5, and at least one of N5 and N6 One (preferably one) combination may be a two-base CpG motif. When the 4-base CpG motif is not located between N3 and N4, in the above formula, any two consecutive bases in the central 4 bases (4th to 7th bases) are CpG motifs, The two bases can be any nucleotide.
　本発明においてより好適に用いられるＫ型ＣｐＧ　ＯＤＮは１つまたは複数のＣｐＧモチーフを含む非回文構造を含有する。更に好適に用いられるＫ型ＣｐＧ　ＯＤＮは１つまたは複数のＣｐＧモチーフを含む非回文構造からなる。 The K-type CpG ODN more preferably used in the present invention contains a non-palindrome structure containing one or more CpG motifs. Further preferably used K-type CpG ODN has a non-palindrome structure containing one or more CpG motifs.
　ヒト化Ｋ型ＣｐＧ　ＯＤＮは、一般的に、ＴＣＧＡ又はＴＣＧＴからなる４塩基のＣｐＧモチーフを特徴とする。また、多くのケースにおいて、一つのヒト化Ｋ型ＣｐＧ　ＯＤＮ中にこの４塩基のＣｐＧモチーフが２又は３個含まれる。従って、好ましい実施形態において、本発明のオリゴデオキシヌクレオチドに含まれるＫ型ＣｐＧ　ＯＤＮは、少なくとも１個、より好ましくは２以上、更に好ましくは２又は３個、ＴＣＧＡ又はＴＣＧＴからなる４塩基のＣｐＧモチーフを含む。該Ｋ型ＣｐＧ　ＯＤＮが２又は３個の４塩基のＣｐＧモチーフを有する場合、これらの４塩基のＣｐＧモチーフは同一であっても異なっていてもよい。ただし、ヒトＴＬＲ９に対するアゴニスト活性を有する限り、特に限定されない。 Humanized K-type CpG ODN is generally characterized by a 4-base CpG motif consisting of TCGA or TCGT. In many cases, two or three of these 4-base CpG motifs are contained in one humanized K-type CpG ODN. Therefore, in a preferred embodiment, the K-type CpG ODN contained in the oligodeoxynucleotide of the present invention has at least one, more preferably two or more, more preferably two or three, a 4-base CpG motif consisting of TCGA or TCGT. including. When the K-type CpG ODN has 2 or 3 4-base CpG motifs, these 4-base CpG motifs may be the same or different. However, there is no particular limitation as long as it has agonist activity against human TLR9.
　本発明のオリゴデオキシヌクレオチドに含まれるＫ型ＣｐＧ　ＯＤＮは、より好ましくは、配列番号１で表されるヌクレオチド配列を含む。 The K-type CpG ODN contained in the oligodeoxynucleotide of the present invention more preferably comprises the nucleotide sequence represented by SEQ ID NO: 1.
　Ｋ型ＣｐＧ　ＯＤＮの長さは、本発明のオリゴデオキシヌクレオチドが免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）を有する限り、特に限定されないが、好ましくは１００ヌクレオチド長以下（例えば、１０−７５ヌクレオチド長）である。Ｋ型ＣｐＧ　ＯＤＮの長さは、より好ましくは５０ヌクレオチド長以下（例えば、１０−４０ヌクレオチド長）である。Ｋ型ＣｐＧ　ＯＤＮの長さは、更に好ましくは３０ヌクレオチド長以下（例えば、１０−２５ヌクレオチド長）である。Ｋ型ＣｐＧ　ＯＤＮの長さは、最も好ましくは、１２−２５ヌクレオチド長である。 The length of K-type CpG ODN is particularly limited as long as the oligodeoxynucleotide of the present invention has immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6). Although not preferred, it is preferably no more than 100 nucleotides long (eg, 10-75 nucleotides long). The length of K-type CpG ODN is more preferably 50 nucleotides or less (for example, 10-40 nucleotides). The length of K-type CpG ODN is more preferably 30 nucleotides or less (for example, 10-25 nucleotides). The length of K-type CpG ODN is most preferably 12-25 nucleotides long.
　ポリデオキシアデニル酸（ｄＡ）の長さは、β−１，３−グルカン（好ましくは、レンチナン、又はシゾフィラン）鎖とともに三重螺旋構造を形成するのに十分な長さであれば特に限定されるものではないが、安定な三重螺旋構造を形成する観点からは、通常２０ヌクレオチド長以上、好ましくは４０ヌクレオチド長以上、より好ましくは６０ヌクレオチド長以上である。ポリｄＡは、長ければ長いほどβ−１，３−グルカンと安定な三重螺旋構造を形成するので、理論的には上限はないが、長すぎると、オリゴデオキシヌクレオチドの合成時の長さにバラつきが生じる原因となるので、通常、１００ヌクレオチド長以下、好ましくは８０以下である。一方、前記安定な三重螺旋構造の形成に加え、単位量のβ−１，３−グルカンあたりに結合する本発明のオリゴデオキシヌクレオチド量を増大させ、且つオリゴデオキシヌクレオチドの合成時の長さのばらつきの回避、複合化効率の観点からは、ポリｄＡの長さは、好ましくは、２０~６０ヌクレオチド長（具体的には、２０，２１，２２，２３，２４，２５，２６，２７，２８，２９，３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０，５１，５２，５３，５４，５５，５６，５７，５８，５９又は６０ヌクレオチド長）、より好ましくは、３０~５０ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０ヌクレオチド長）１８、最も好ましくは、３０~４５ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５ヌクレオチド長）である。特に、３０ヌクレオチド長以上の場合、良好な複合化効率を示す。本発明のオリゴデオキシヌクレオチドは、ポリｄＡを含むことにより、２本のシゾフィラン鎖とともに三重螺旋構造を形成する活性を有する。なお、ポリデオキシアデニル酸を「ポリ（ｄＡ）」又は「ｐｏｌｙ（ｄＡ）」と表記することもある。 The length of polydeoxyadenylic acid (dA) is particularly limited as long as it is long enough to form a triple helical structure with a β-1,3-glucan (preferably lentinan or schizophyllan) chain. However, from the viewpoint of forming a stable triple helix structure, it is usually 20 nucleotides or more, preferably 40 nucleotides or more, more preferably 60 nucleotides or more. Poly dA forms a stable triple helix structure with β-1,3-glucan the longer it is, so there is theoretically no upper limit, but if it is too long, the length at the time of synthesis of oligodeoxynucleotides will vary. Is usually 100 nucleotides or less, preferably 80 or less. On the other hand, in addition to the formation of the stable triple helix structure, the amount of the oligodeoxynucleotide of the present invention bound per unit amount of β-1,3-glucan is increased, and the length variation during the synthesis of the oligodeoxynucleotide From the viewpoint of avoiding the above and the efficiency of conjugation, the length of poly dA is preferably 20 to 60 nucleotides (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides long), more preferably 30 to 50 nucleotides long (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) , 42 , 43, 44, 45, 46, 47, 48, 49, 50 nucleotides) 18, most preferably 30-45 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39). , 40, 41, 42, 43, 44, 45 nucleotides long). In particular, when the length is 30 nucleotides or more, good conjugation efficiency is exhibited. The oligodeoxynucleotide of the present invention has an activity of forming a triple helical structure with two schizophyllan chains by including poly dA. In addition, polydeoxyadenylic acid may be described as “poly (dA)” or “poly (dA)”.
　１分子の本発明のオリゴデオキシヌクレオチドには、複数個のＫ型ＣｐＧ　ＯＤＮ及び／又はポリｄＡが含まれていてもよいが、好ましくは、Ｋ型ＣｐＧ　ＯＤＮ及ポリｄＡが１つずつ含まれ、最も好ましくは、Ｋ型ＣｐＧ　ＯＤＮ及ポリｄＡが１つずつからなる。 One molecule of the oligodeoxynucleotide of the present invention may contain a plurality of K-type CpG ODN and / or poly dA, but preferably contains one K-type CpG ODN and poly dA one by one, Most preferably, each of K-type CpG ODN and poly dA consists of one by one.
　例示的なＣｐＧの配列としては、Ｋ３　ＣｐＧ（配列番号１＝５’−ａｔｃｇａｃｔｃｔｃｇａｇｃｇｔｔｃｔｃ−３’）等を挙げることができるがこれに限定されない。 Examples of CpG sequences include, but are not limited to, K3 CpG (SEQ ID NO: 1 = 5'-atcgactctcgagcgttctc-3 ').
　本発明のオリゴデオキシヌクレオチドは、ポリｄＡがＫ型ＣｐＧ　ＯＤＮの３’側に配置されていることを特徴とする。この配置により、本発明の複合体（詳細は下に述べる）が、抗がん作用も増強される可能性があるものと思われるがこれらに限定されず、抗がん剤としてはいずれに結合させてもよい。 The oligodeoxynucleotide of the present invention is characterized in that poly dA is arranged on the 3 'side of K-type CpG ODN. With this arrangement, it is considered that the complex of the present invention (details will be described below) may also enhance the anticancer activity, but the present invention is not limited thereto. You may let them.
　Ｋ型ＣｐＧ　ＯＤＮとポリｄＡとは、直接共有結合により連結されていてもよいし、スペーサー配列を介して連結されていてもよい。スペーサー配列とは、２つの近接した構成要素の間に挿入される１以上のヌクレオチドを含むヌクレオチド配列を意味する。スペーサー配列の長さは、本発明の複合体が、免疫賦活活性（好ましくはＢ細胞を活性化してＩＬ−６を産生させる活性、及び樹状細胞を活性化してＩＦＮ−αを産生させる活性）を有する限り、特に限定されないが、通常１~１０ヌクレオチド長、好ましくは１~５ヌクレオチド長、より好ましくは１~３ヌクレオチド長である。最も好ましくは、Ｋ型ＣｐＧ　ＯＤＮとポリｄＡとが、直接共有結合により連結される。 The K-type CpG ODN and poly dA may be directly linked by a covalent bond or may be linked via a spacer sequence. By spacer sequence is meant a nucleotide sequence comprising one or more nucleotides inserted between two adjacent components. The length of the spacer sequence is such that the complex of the present invention has an immunostimulatory activity (preferably an activity to activate B cells to produce IL-6 and an activity to activate dendritic cells to produce IFN-α). However, it is usually 1 to 10 nucleotides long, preferably 1 to 5 nucleotides long, more preferably 1 to 3 nucleotides long. Most preferably, K-type CpG ODN and poly dA are linked by a direct covalent bond.
　本発明のオリゴデオキシヌクレオチドは、Ｋ型ＣｐＧ　ＯＤＮ、ポリｄＡ及び任意的なスペーサー配列に加え、その５’末端及び／又は３’末端に付加的なヌクレオチド配列を有していてもよい。該付加的なヌクレオチド配列の長さは、本発明の複合体が免疫賦活活性（好ましくはＢ細胞を活性化してＩＬ−６を産生させる活性、及び樹状細胞を活性化してＩＦＮ−αを産生させる活性）を有する限り、特に限定されないが、通常１~１０ヌクレオチド長、好ましくは１~５ヌクレオチド長、より好ましくは１~３ヌクレオチド長である。 The oligodeoxynucleotide of the present invention may have an additional nucleotide sequence at its 5 'end and / or 3' end in addition to K-type CpG ODN, poly dA and an optional spacer sequence. The length of the additional nucleotide sequence indicates that the complex of the present invention has an immunostimulatory activity (preferably an activity to activate B cells to produce IL-6, and a dendritic cell to produce IFN-α. However, the length is usually 1 to 10 nucleotides, preferably 1 to 5 nucleotides, more preferably 1 to 3 nucleotides.
　好ましい態様において、本発明のオリゴデオキシヌクレオチドは、このような５’末端及び／又は３’末端の付加的なヌクレオチド配列を含まない。即ち、本発明のオリゴデオキシヌクレオチドは、好ましくは、Ｋ型ＣｐＧ　ＯＤＮ、ポリｄＡ及び任意的なスペーサー配列からなり、更に好ましくは、Ｋ型ＣｐＧ　ＯＤＮ及びポリｄＡからなる。 In a preferred embodiment, the oligodeoxynucleotide of the present invention does not contain such additional nucleotide sequences at the 5 'end and / or the 3' end. That is, the oligodeoxynucleotide of the present invention preferably comprises K-type CpG ODN, poly dA and an optional spacer sequence, and more preferably comprises K-type CpG ODN and poly dA.
　最も好ましい態様において、本発明のオリゴデオキシヌクレオチドは、Ｋ型ＣｐＧ　ＯＤＮ（具体的には、例えば、配列番号１で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチド）及びポリｄＡからなり、Ｋ型ＣｐＧ　ＯＤＮが該オリゴデオキシヌクレオチドの５’末端に、ポリｄＡが３’末端に、それぞれ位置する。具体的には、配列番号１で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチドの３’末端に２０~６０ヌクレオチド長（より好ましくは、３０~５０ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０ヌクレオチド長）、最も好ましくは、３０~４５ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５ヌクレオチド長））のポリｄＡが結合したオリゴデオキシヌクレオチドであり、例えば、配列番号２、又は９~１２で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチドである。 In the most preferred embodiment, the oligodeoxynucleotide of the present invention comprises K-type CpG ODN (specifically, for example, an oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1) and poly dA, and comprises K-type CpG ODN. Is located at the 5 ′ end of the oligodeoxynucleotide and poly dA at the 3 ′ end. Specifically, the oligodeoxynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleotides (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long), most preferably 30-45 nucleotides long (30, 31, 32) , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides))), which is an oligodeoxynucleotide bound to, for example, SEQ ID NO: 2 or 9 It is an oligodeoxynucleotide consisting of the nucleotide sequence represented by ~ 12.
　本発明のオリゴデオキシヌクレオチドの全長は、通常３０~２００ヌクレオチド長、好ましくは３５~１００ヌクレオチド長、より好ましくは４０~８０ヌクレオチド長（具体的には、４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０，５１，５２，５３，５４，５５，５６，５７，５８，５９，６０，６１，６２，６３，６４，６５，６６，６７，６８，６９，７０，７１，７２，７３，７４，７５，７６，７７，７８，７９又は８０ヌクレオチド長）、より好ましくは、５０~７０ヌクレオチド長（具体的には、５０，５１，５２，５３，５４，５５，５６，５７，５８，５９，６０，６１，６２，６３，６４，６５，６６，６７，６８，６９，７０ヌクレオチド長）、最も好ましくは、５０~６５ヌクレオチド長（具体的には、５０，５１，５２，５３，５４，５５，５６，５７，５８，５９，６０，６１，６２，６３，６４，６５ヌクレオチド長）である。　本発明のオリゴデオキシヌクレオチドは、インビボにおける分解（例、エクソ又はエンドヌクレアーゼによる分解）に対して抵抗性となるように適切に修飾されていてもよい。好ましくは、該改変はホスホロチオエート修飾又はホスホロジチオエート修飾を含む。即ち、本発明のオリゴデオキシヌクレオチド中のリン酸ジエステル結合の一部又は全てが、ホスホロチオエート結合又はホスホロジチオエート結合により置換されている。 The total length of the oligodeoxynucleotide of the present invention is generally 30 to 200 nucleotides, preferably 35 to 100 nucleotides, more preferably 40 to 80 nucleotides (specifically, 40, 41, 42, 43, 44, 45). , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides long), more preferably 50 to 70 nucleotides long (specifically, 50, 51, 52, 53, 54, 55). , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 nucleotides), most preferably 50 to 65 nucleotides Thereof include a 50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65 nucleotides in length). The oligodeoxynucleotide of the present invention may be appropriately modified so as to be resistant to in vivo degradation (eg, degradation by exo or endonuclease). Preferably, the modification comprises a phosphorothioate modification or a phosphorodithioate modification. That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is substituted by a phosphorothioate bond or a phosphorodithioate bond.
　好ましくは、本発明のオリゴデオキシヌクレオチドは、リン酸ジエステル結合の修飾を含み、より好ましくは、リン酸ジエステル結合の修飾は、ホスホロチオエート結合（即ち、ＷＯ　９５／２６２０４に記載されているように、非架橋酸素原子のうちの１つが、硫黄原子に置換される）である。即ち、本発明のオリゴデオキシヌクレオチド中のリン酸ジエステル結合の一部又は全てが、ホスホロチオエート結合により置換されている。 Preferably, the oligodeoxynucleotide of the present invention comprises a modification of a phosphodiester bond, more preferably the modification of a phosphodiester bond is a non-phosphorothioate bond (ie, as described in WO 95/26204). One of the bridging oxygen atoms is replaced by a sulfur atom). That is, part or all of the phosphodiester bond in the oligodeoxynucleotide of the present invention is replaced by a phosphorothioate bond.
　本発明のオリゴデオキシヌクレオチドは、好ましくは、Ｋ型ＣｐＧ　ＯＤＮにおいて、ホスホロチオエート結合、または、ホスホロジチオエート結合による修飾を含み、より好ましくは、該Ｋ型ＣｐＧ　ＯＤＮのリン酸ジエステル結合の全てが、ホスホロチオエート結合に置換される。また、本発明のオリゴデオキシヌクレオチドは、好ましくは、ポリｄＡにおいて、ホスホロチオエート結合、または、ホスホロジチオエート結合を含み、より好ましくは、該ポリｄＡのリン酸ジエステル結合の全てが、ホスホロチオエート結合に置換される。更に好ましくは、本発明のヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含むオリゴデオキシヌクレオチドのリン酸ジエステル結合の全てが、ホスホロチオエート結合に置換される。最も好ましくは、本発明のオリゴデオキシヌクレオチドは、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド（例、配列番号１）の３’末端に２０~６０ヌクレオチド長（より好ましくは、３０~５０ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０ヌクレオチド長）、最も好ましくは、３０~４５ヌクレオチド長（３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５ヌクレオチド長））のポリｄＡが結合したオリゴデオキシヌクレオチドであって、当該オリゴデオキシヌクレオチドに含まれる全てのリン酸ジエステル結合が、ホスホロチオエート結合に置換されている。ホスホロチオエート結合により、本発明のオリゴデオキシヌクレオチドにおいて、分解に対する抵抗性のみならず、免疫賦活活性（例えば、ｐＤＣを活性化させてＩＦＮ−αを産生させる活性）の増強、及びＣｐＧ−β−１，３−グルカン複合体の高収率、ならびに抗がん活性の増強が期待されるからである。なお、本明細書におけるホスホロチオエート結合はホスホロチオエート骨格と、リン酸ジエステル結合はリン酸骨格と同義である。本発明のオリゴデオキシヌクレオチドには、上記オリゴデオキシヌクレオチドのあらゆる薬学的に許容可能な塩類、エステル、またはそのようなエステルの塩類を含む。 The oligodeoxynucleotide of the present invention preferably contains a modification by phosphorothioate linkage or phosphorodithioate linkage in K-type CpG ODN, more preferably all of the phosphodiester linkage of K-type CpG ODN is Substituted with a phosphorothioate linkage. The oligodeoxynucleotide of the present invention preferably contains a phosphorothioate bond or a phosphorodithioate bond in poly dA, and more preferably all of the phosphodiester bonds of poly dA are replaced with phosphorothioate bonds. Is done. More preferably, all of the phosphodiester linkages of oligodeoxynucleotides, including humanized K-type CpG oligodeoxynucleotides and polydeoxyadenylates of the invention, are replaced with phosphorothioate linkages. Most preferably, the oligodeoxynucleotide of the present invention has a length of 20 to 60 nucleotides (more preferably 30 to 50 nucleotides (30, 30) at the 3 ′ end of a humanized K-type CpG oligodeoxynucleotide (eg, SEQ ID NO: 1). 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides), most preferably 30-45 An oligodeoxynucleotide having a nucleotide length (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides) bound thereto , All phosphodiester bonds contained in the oligodeoxynucleotide are substituted with phosphorothioate bonds. By the phosphorothioate bond, in the oligodeoxynucleotide of the present invention, not only the resistance to degradation but also the immunostimulatory activity (for example, the activity of activating pDC to produce IFN-α), and CpG-β-1, This is because high yield of 3-glucan complex and enhancement of anticancer activity are expected. In the present specification, the phosphorothioate bond is synonymous with the phosphorothioate skeleton, and the phosphate diester bond is synonymous with the phosphate skeleton. The oligodeoxynucleotides of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters of the above oligodeoxynucleotides.
　本発明のオリゴデオキシヌクレオチドの薬学的に許容可能な塩類としては、好適にはナトリウム塩、カリウム塩、リチウム塩のようなアルカリ金属塩、カルシウム塩、マグネシウム塩のようなアルカリ土類金属塩、アルミニウム塩、鉄塩、亜鉛塩、銅塩、ニッケル塩、コバルト塩等の金属塩；アンモニウム塩のような無機塩、ｔ−オクチルアミン塩、ジベンジルアミン塩、モルホリン塩、グルコサミン塩、フェニルグリシンアルキルエステル塩、エチレンジアミン塩、Ｎ−メチルグルカミン塩、グアニジン塩、ジエチルアミン塩、トリエチルアミン塩、ジシクロヘキシルアミン塩、Ｎ，Ｎ’−ジベンジルエチレンジアミン塩、クロロプロカイン塩、プロカイン塩、ジエタノールアミン塩、Ｎ−ベンジル−フェネチルアミン塩、ピペラジン塩、テトラメチルアンモニウム塩、トリス（ヒドロキシメチル）アミノメタン塩のような有機塩等のアミン塩；弗化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩のようなハロゲン原子化水素酸塩、硝酸塩、過塩素酸塩、硫酸塩、リン酸塩等の無機酸塩；メタンスルホン酸塩、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩のような低級アルカンスルホン酸塩、ベンゼンスルホン酸塩、ｐ−トルエンスルホン酸塩のようなアリ−ルスルホン酸塩、酢酸塩、リンゴ酸塩、フマ−ル酸塩、コハク酸塩、クエン酸塩、酒石酸塩、蓚酸塩、マレイン酸塩等の有機酸塩；及び、グリシン塩、リジン塩、アルギニン塩、オルニチン塩、グルタミン酸塩、アスパラギン酸塩のようなアミノ酸塩を挙げることができる。 The pharmaceutically acceptable salts of the oligodeoxynucleotide of the present invention are preferably alkali metal salts such as sodium salt, potassium salt and lithium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum Metal salts such as salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl esters Salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine Salt, piperazine salt Amine salts such as organic salts such as tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; Halogenated hydrogen halides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide Inorganic acid salts such as acid salts, nitrates, perchlorates, sulfates, phosphates; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate Organic acids such as aryl sulfonates such as p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate And amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, aspartate.
　本発明のオリゴデオキシヌクレオチドは、１本鎖、２本鎖、３本鎖のいずれの形態でもよいが、好ましくは１本鎖である。 The oligodeoxynucleotide of the present invention may be in any form of single strand, double strand, and triple strand, but is preferably single strand.
　本発明のオリゴデオキシヌクレオチドは、好ましくは単離されている。「単離」とは、目的とする成分以外の因子を除去する操作がなされ、天然に存在する状態を脱していることを意味する。「単離されたオリゴデオキシヌクレオチド」の純度（評価対象物の総重量に占める目的とするオリゴデオキシヌクレオチド重量の百分率）は、通常７０％以上、好ましくは８０％以上、より好ましくは９０％以上、更に好ましくは９９％以上である。 The oligodeoxynucleotide of the present invention is preferably isolated. “Isolated” means that an operation to remove factors other than the target component has been performed, and that the naturally occurring state has been removed. The purity of the “isolated oligodeoxynucleotide” (percentage of the desired oligodeoxynucleotide weight in the total weight of the evaluation object) is usually 70% or more, preferably 80% or more, more preferably 90% or more, More preferably, it is 99% or more.
　本発明のオリゴデオキシヌクレオチドは、優れた免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）を有するので、免疫賦活剤等として有用である。更に、本発明のオリゴデオキシヌクレオチドは、２本のβ−１，３−グルカン（好ましくは、シゾフィラン、レンチナン又はスクレログルカン）とともに三重螺旋構造を形成する性質を有するので、本発明の複合体の調製に有用である。 The oligodeoxynucleotide of the present invention has excellent immunostimulatory activity (for example, the activity of activating B cells (preferably human B cells) to produce IL-6), and thus is useful as an immunostimulator or the like. . Furthermore, since the oligodeoxynucleotide of the present invention has a property of forming a triple helix structure with two β-1,3-glucans (preferably, schizophyllan, lentinan or scleroglucan), the complex of the present invention Useful for preparation.
　本発明は、上記本発明のオリゴデオキシヌクレオチド及びβ−１，３−グルカンを含有する複合体（以下、本発明の複合体と称する。）を提供するものである。 The present invention provides a complex containing the oligodeoxynucleotide of the present invention and β-1,3-glucan (hereinafter referred to as the complex of the present invention).
　上述の本発明のオリゴデオキシヌクレオチドは、Ｋ型ＣｐＧ　ＯＤＮを含むので、それ単独では、Ｋ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）を発揮し、Ｄ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、形質細胞様樹状細胞を活性化してＩＦＮ−αを産生させる活性）に乏しい。しかしながら、驚くべきことに、β−１，３−グルカン（好ましくは、レンチナン、シゾフィラン）と複合体を形成することにより、Ｄ型ＣｐＧ　ＯＤＮの配列を要することなく、Ｄ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、形質細胞様樹状細胞を活性化してＩＦＮ−αを産生させる活性）を獲得する。即ち、本発明の複合体は、Ｋ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）と、Ｄ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、形質細胞様樹状細胞（好ましくはヒト形質細胞様樹状細胞）を活性化してＩＦＮ−αを産生させる活性）の両方を有する。本発明で用いられるβ−１，３−グルカンとしては、シゾフィラン、スクレログルカン、カードラン、パーキマン、グリホラン、レンチナン、ラミナラン等を挙げることが出来る。β−１，３−グルカンは、好ましくは、シゾフィラン、レンチナンまたはスクレログルカンのように、１，６−グルコピラノシド分枝を多く含有する（側鎖率３３~４０％）β−１，３−グルカンであり、より好ましくはシゾフィランである。 Since the above-mentioned oligodeoxynucleotide of the present invention contains K-type CpG ODN, it alone activates an immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells) and IL -6), and immunostimulatory activity peculiar to D-type CpG ODN (for example, the activity of activating plasmacytoid dendritic cells to produce IFN-α) is poor. However, surprisingly, by forming a complex with β-1,3-glucan (preferably lentinan, schizophyllan), the immunity specific to D-type CpG ODN is not required, without requiring a D-type CpG ODN sequence. An activation activity (for example, an activity of activating plasmacytoid dendritic cells to produce IFN-α) is acquired. That is, the complex of the present invention has an immunostimulatory activity peculiar to K-type CpG ODN (for example, an activity that activates B cells (preferably human B cells) to produce IL-6) and D-type CpG ODN. Specific immunostimulatory activity (for example, the activity of activating plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) to produce IFN-α). Examples of β-1,3-glucan used in the present invention include schizophyllan, scleroglucan, curdlan, parkan, glyphoran, lentinan, laminaran and the like. The β-1,3-glucan preferably contains a large amount of 1,6-glucopyranoside branches (side chain ratio 33 to 40%), such as schizophyllan, lentinan or scleroglucan. More preferred is schizophyllan.
　レンチナン（ＬＮＴ）は、シイタケ由来の公知のβ−１，３−１，６−グルカンであり、分子式は（Ｃ６Ｈ１００５）ｎ、分子量は約３０~７０万である。水、メタノール、エタノール（９５）、又はアセトンにはほとんど溶けないが、極性有機溶媒であるＤＭＳＯや水酸化ナトリウム水溶液に溶解する。 Lentinan (LNT) is a known β-1,3-1,6-glucan derived from Shiitake mushroom, has a molecular formula of (C6H1005) n, and a molecular weight of about 300,000 to 700,000. It hardly dissolves in water, methanol, ethanol (95), or acetone, but dissolves in DMSO or sodium hydroxide aqueous solution, which are polar organic solvents.
　レンチナンは活性化マクロファージ、キラーＴ細胞、ナチュラルキラー細胞及び抗体依存性マクロファージ仲介性細胞障害作用（ＡＤＭＣ）活性の増強作用を有する（Ｈａｍｕｒｏ，Ｊ．，ｅｔ　ａｌ．：Ｉｍｍｕｎｏｌｏｇｙ，３９，５５１−５５９，１９８０、Ｈａｍｕｒｏ，Ｊ．，ｅｔ　ａｌ．：Ｉｎｔ．Ｊ．Ｉｍｍｕｎｏｐｈａｒｍａｃｏｌ．，２，１７１，１９８０、Ｈｅｒｌｙｎ，Ｄ．，ｅｔ　ａｌ．：Ｇａｎｎ，７６，３７−４２，１９８５）。動物実験においては同系腫瘍及び自家腫瘍に対して化学療法剤との併用投与により腫瘍増殖抑制作用ならびに延命効果が認められている。また、レンチナンの単独投与によっても腫瘍増殖抑制作用ならびに延命効果が認められている。臨床試験においては手術不能又は再発胃癌患者に対して、テガフール経口投与との併用により生存期間の延長が認められ（医薬品インタビューフォーム「レンチナン静注用１ｍｇ「味の素」」）、本邦で承認されている。レンチナンの単独投与による効果は現在のところ確認されていない。 Lentinan has an effect of enhancing activated macrophages, killer T cells, natural killer cells and antibody-dependent macrophage-mediated cytotoxicity (ADMC) activity (Hamuro, J., et al .: Immunology, 39, 551-559, 1980, Hamuro, J., et al .: Int. J. Immunopharmacol., 2, 171, 1980, Herlyn, D., et al .: Gann, 76, 37-42, 1985). In animal experiments, tumor growth suppression and life-prolonging effects have been observed for syngeneic tumors and autologous tumors by combined administration with chemotherapeutic agents. In addition, administration of lentinan alone has been shown to suppress tumor growth and prolong life. In clinical trials, patients with inoperable or recurrent gastric cancer have prolonged survival when combined with oral administration of tegafur (pharmaceutical interview form "1 mg lentinan for intravenous injection Ajinomoto") and has been approved in Japan. . The effect of single administration of lentinan has not been confirmed so far.
　シゾフィラン（ＳＰＧ）は、スエヒロタケ由来の公知の可溶性β−グルカンである。ＳＰＧは、β−（１→３）−Ｄ−グルカンの主鎖と、各３個のグルコース当り１個のβ−（１→６）−Ｄ−グルコシル側鎖からなる（Ｔａｂａｔａ，Ｋ．，Ｉｔｏ，Ｗ．，Ｋｏｊｉｍａ，Ｔ．，Ｋａｗａｂａｔａ，Ｓ．ａｎｄ　Ｍｉｓａｋｉ　Ａ．，「Ｃａｒｂｏｈｙｄｒ．Ｒｅｓ．」，１９８１，８９，１，ｐ．１２１−１３５）。ＳＰＧは婦人科癌に対する免疫増強法の筋肉内注射製剤臨床薬として２０年以上の使用実績があり（清水，陳，荷見，増淵，「Ｂｉｏｔｈｅｒａｐｙ」，１９９０，４，ｐ．１３９０長谷川，「Ｏｎｃｏｌｏｇｙ　ａｎｄ　Ｃｈｅｍｏｔｈｅｒａｐｙ」，１９９２，８，ｐ．２２５）、生体内での安全性が確認されている（Ｔｈｅｒｅｓａ，Ｍ．ＭｃＩｎｔｉｒｅ　ａｎｄ　Ｄａｖｉｄ，Ａ．Ｂｒａｎｔ，「Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．」，１９９８，１２０，ｐ．６９０９）。 Schizophyllan (SPG) is a known soluble β-glucan derived from Shirohirotake. SPG consists of a β- (1 → 3) -D-glucan main chain and one β- (1 → 6) -D-glucosyl side chain for each three glucoses (Tabata, K., Ito). , W., Kojima, T., Kawabata, S. and Misaki A., “Carbohydr. Res.”, 1981, 89, 1, p. SPG has been used for more than 20 years as a clinical drug for intramuscular injection of immunity enhancement method against gynecological cancer (Shimizu, Chen, Kazumi, Masumi, “Biotherapy”, 1990, 4, p. 1390 Hasegawa, “Oncology”. and Chemotherapy ”, 1992, 8, p. 225), in-vivo safety has been confirmed (Theresa, M. McIntire and David, A. Brant,“ J. Am. Chem. Soc. ”, 1998, 120, p.6909).
　本明細書において「複合体」とは、複数の分子が、静電結合、ファンデルワールス結合、水素結合、疎水性相互作用などの非共有結合又は共有結合を介して会合することにより得られる産物を意味する。 As used herein, “complex” refers to a product obtained by association of a plurality of molecules through non-covalent or covalent bonds such as electrostatic bonds, van der Waals bonds, hydrogen bonds, and hydrophobic interactions. Means.
　本発明の複合体は、好ましくは、三重螺旋構造状である。好ましい態様において、当該三重螺旋構造を形成する３本の鎖のうち、２本はβ−１，３−グルカン鎖であり、１本は、上記本発明のオリゴデオキシヌクレオチド中のポリデオキシアデニル酸の鎖である。なお、当該複合体は一部に、三重螺旋構造を形成していない部分を含んでいても良い。 The complex of the present invention preferably has a triple helical structure. In a preferred embodiment, two of the three chains forming the triple helix structure are β-1,3-glucan chains, and one is the polydeoxyadenylic acid in the oligodeoxynucleotide of the present invention. Is a chain. Note that the complex may partially include a portion that does not form a triple helical structure.
　本発明の複合体における、オリゴデオキシヌクレオチドとβ−１，３−グルカンの組成比は、オリゴデオキシヌクレオチド中のポリデオキシアデニル酸の鎖長、及びβ−１，３−グルカンの長さ等に応じて、変化しうる。例えば、β−１，３−グルカン鎖と、ポリデオキシアデニル酸の鎖の長さが同等の場合には、２本のβ−１，３−グルカン鎖と、１本の本発明のオリゴデオキシヌクレオチドが会合し、三重螺旋構造を形成し得る。一般的には、β−１，３−グルカン鎖に対して、ポリデオキシアデニル酸の鎖長は短いので、２本のβ−１，３−グルカン鎖に対して、複数の本発明のオリゴデオキシヌクレオチドがポリデオキシアデニル酸を介して会合し、三重螺旋構造を形成し得る（図１参照）。 The composition ratio of oligodeoxynucleotide and β-1,3-glucan in the complex of the present invention depends on the chain length of polydeoxyadenylic acid in the oligodeoxynucleotide, the length of β-1,3-glucan, etc. Can change. For example, when the lengths of β-1,3-glucan chains and polydeoxyadenylic acid chains are equivalent, two β-1,3-glucan chains and one oligodeoxynucleotide of the present invention Can associate to form a triple helix structure. In general, since the chain length of polydeoxyadenylic acid is shorter than that of β-1,3-glucan chains, a plurality of oligodeoxy compounds of the present invention are used for two β-1,3-glucan chains. Nucleotides can associate through polydeoxyadenylate to form a triple helix structure (see FIG. 1).
　本発明の複合体は、ヒト化Ｋ型ＣｐＧ　ＯＤＮ及びβ−１，３−グルカン（例、レンチナン、シゾフィラン、スクレログルカン、カードラン、パーキマン、グリホラン、ラミナラン）を含有する複合体であり、好ましくは、ヒト化Ｋ型ＣｐＧ　ＯＤＮ及びβ−１，３−グルカン（例、レンチナン、シゾフィラン、スクレログルカン）からなる複合体である。より好ましくは、配列番号１で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチドの３’側に２０~６０ヌクレオチド長（具体的には、２０，２１，２２，２３，２４，２５，２６，２７，２８，２９，３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０，５１，５２，５３，５４，５５，５６，５７，５８，５９又は６０ヌクレオチド長）のポリデオキシアデニル酸が結合し、かつリン酸ジエステル結合の全てがホスホロチオエート結合に置換されたオリゴデオキシヌクレオチド、及びβ−１，３−グルカン（例、レンチナン、シゾフィラン）からなる複合体（例、Ｋ３−ｄＡ２０~６０−ＬＮＴ、Ｋ３−ｄＡ２０~６０−ＳＰＧ）であり、更に好ましくは、配列番号１で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチドの３’側に３０~５０ヌクレオチド長（具体的には、３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８，４９，５０ヌクレオチド長）のポリデオキシアデニル酸が結合し、かつリン酸ジエステル結合の全てがホスホロチオエート結合に置換されたオリゴデオキシヌクレオチド、及びβ−１，３−グルカン（例、レンチナン、シゾフィラン）からなる複合体（例、Ｋ３−ｄＡ３０~５０−ＬＮＴ、Ｋ３−ｄＡ３０~５０−ＳＰＧ）であり、最も好ましくは、配列番号１で表されるヌクレオチド配列からなるオリゴデオキシヌクレオチドの３’側に３０~４５ヌクレオチド長（具体的には、３０，３１，３２，３３，３４，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５ヌクレオチド長）のポリデオキシアデニル酸が結合し、かつリン酸ジエステル結合の全てがホスホロチオエート結合に置換されたオリゴデオキシヌクレオチド、及びβ−１，３−グルカン（例、レンチナン、シゾフィラン）からなる複合体（Ｋ３−ｄＡ３０~４５−ＬＮＴ、Ｋ３−ｄＡ３０~４５−ＳＰＧ）である。 The complex of the present invention is a complex containing humanized K-type CpG ODN and β-1,3-glucan (eg, lentinan, schizophyllan, scleroglucan, curdlan, perchiman, glyphoran, laminaran), preferably Is a complex consisting of humanized K-type CpG ODN and β-1,3-glucan (eg, lentinan, schizophyllan, scleroglucan). More preferably, the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a length of 20 to 60 nucleotides on the 3 ′ side (specifically, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length), and oligodeoxynucleotides in which all of the phosphodiester bonds are replaced with phosphorothioate bonds, and β-1, A complex composed of 3-glucan (eg, lentinan, schizophyllan) (eg, K3-dA20-60-LNT, K3-dA20-60-SPG); Preferably, the oligodeoxynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 1 has a length of 30 to 50 nucleotides on the 3 ′ side (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides long) and all of the phosphodiester bonds were replaced with phosphorothioate bonds. A complex (eg, K3-dA30-50-LNT, K3-dA30-50-SPG) composed of an oligodeoxynucleotide and β-1,3-glucan (eg, lentinan, schizophyllan), most preferably a sequence The oligodeoxynucleotide consisting of the nucleotide sequence represented by No. 1 has a length of 30 to 45 nucleotides on the 3 ′ side (specifically, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 nucleotides long) and deoxyphosphate bond A complex (K3-dA30-45-LNT, K3-dA30-45-SPG) consisting of an oligodeoxynucleotide substituted with a phosphorothioate bond and a β-1,3-glucan (eg, lentinan, schizophyllan) is there.
　本発明の複合体の調製方法に関しては、非特許文献２１~２４や、特開２００８−１００９１９号公報に記載された条件と同様に行うことができる。すなわち、本来は、天然で三重螺旋構造として存在するβ−１，３−グルカンを非プロトン性有機極性溶媒（ジメチルスルホキシド（ＤＭＳＯ），アセトニトリル，アセトン等）またはアルカリ水溶液（水酸化ナトリウム、水酸化カリウム、アンモニア、水酸化カルシウム等）に溶解して一本鎖に解く。このようにして得られた一本鎖のβ−１，３−グルカンの溶液と本発明のオリゴデオキシヌクレオチドの溶液（水溶液、中性付近のｐＨの緩衝水溶液、又は酸性の緩衝水溶液、好ましくは、水溶液又は中性付近のｐＨの緩衝水溶液）とを混合し、必要に応じて再度ｐＨを中性付近に調整後、適当時間保持する、例えば、５℃で一夜保持する。その結果、２本のβ−１，３−グルカン鎖と該オリゴデオキシヌクレオチド中のポリｄＡ鎖が三重螺旋構造を形成することにより、本発明の複合体が形成される。生成した複合体に対して、サイズ排除クロマトグラフィーによる精製、限外濾過、透析等を行うことにより、複合体未形成のオリゴデオキシヌクレオチドを除くことができる。また、生成した複合体に対して、陰イオン交換クロマトグラフィーによる精製を行うことにより、複合体未形成のβ−１，３−グルカンを除くことができる。上記の方法により、複合体を適宜精製することができる。 The method for preparing the complex of the present invention can be carried out in the same manner as the conditions described in Non-Patent Documents 21 to 24 and Japanese Patent Application Laid-Open No. 2008-1000091. That is, β-1,3-glucan, which originally exists as a triple helical structure in nature, is converted into an aprotic organic polar solvent (dimethyl sulfoxide (DMSO), acetonitrile, acetone, etc.) or an alkaline aqueous solution (sodium hydroxide, potassium hydroxide). , Ammonia, calcium hydroxide, etc.) The solution of the single-stranded β-1,3-glucan thus obtained and the oligodeoxynucleotide solution of the present invention (aqueous solution, buffer solution having a pH near neutral, or acidic buffer solution, preferably Aqueous solution or a buffered aqueous solution having a pH near neutral), and after adjusting the pH again to near neutral as necessary, hold it for an appropriate time, for example, hold it at 5 ° C. overnight. As a result, the complex of the present invention is formed by the two β-1,3-glucan chains and the poly dA chain in the oligodeoxynucleotide forming a triple helical structure. By performing purification by size exclusion chromatography, ultrafiltration, dialysis and the like on the produced complex, oligodeoxynucleotides not formed in the complex can be removed. Further, by performing purification by anion exchange chromatography on the produced complex, β-1,3-glucan having no complex formed can be removed. By the above method, the complex can be appropriately purified.
　本発明の複合体の形成は、例えばＣＤ（円偏光二色性）スペクトルによるコンフォメーション変化、サイズ排除クロマトグラフィーによるＵＶ吸収シフト、ゲル電気泳動、マイクロチップ電気泳動、キャピラリー電気泳動を測定することにより確認することができるが、これに限らない。 The formation of the complex of the present invention is performed by measuring, for example, conformational change by CD (circular dichroism) spectrum, UV absorption shift by size exclusion chromatography, gel electrophoresis, microchip electrophoresis, capillary electrophoresis. Although it can confirm, it is not restricted to this.
　本発明のオリゴデオキシヌクレオチドとβ−１，３−グルカンとの混合比は、ポリｄＡ鎖の長さ等を考慮して適宜設定することができるが、通常モル比（ＳＰＧ／ＯＤＮ）が０．０２~２．０、好ましくは０．１~０．５である。更なる態様において、モル比（β−１，３−グルカン（ＬＮＴ等）／ＯＤＮ）が例えば０．００５~１．０、好ましくは０．０２０~０．２５である。 The mixing ratio of the oligodeoxynucleotide of the present invention and β-1,3-glucan can be appropriately set in consideration of the length of the poly dA chain and the like, but usually the molar ratio (SPG / ODN) is 0. It is 02 to 2.0, preferably 0.1 to 0.5. In a further embodiment, the molar ratio (β-1,3-glucan (LNT, etc.) / ODN) is, for example, 0.005 to 1.0, preferably 0.020 to 0.25.
　本発明の複合体の調製方法についてＣｐＧ−ＯＤＮとＬＮＴ複合体を例に説明する。ＬＮＴを０．０５~２Ｎ、好ましくは０．１~１．５Ｎのアルカリ水溶液（例えば、０．２５Ｎ水酸化ナトリウム水溶液）に溶解させ、１℃~４０℃で１０時間~４日間放置し（例えば、室温で一晩放置する）、一本鎖のＬＮＴ水溶液（例えば、５０ｍｇ／ｍｌのＬＮＴ水溶液）を調製する。前記ＬＮＴ水溶液と別途調製したＣｐＧ水溶液（例えば、１００μＭのＣｐＧ水溶液）をモル比（ＬＮＴ／ＯＤＮ）０．００５~１．０で混合し、続いて、前記ＬＮＴ水溶液に酸性の緩衝水溶液（例えば、ＮａＨ２ＰＯ４）を加えて中和し、１~４０℃で６時間~４日間（例えば、４℃で一晩）維持することで複合化を完了させる。なお、前記複合化のためにＬＮＴ水溶液を最後に加え混合しても良い。複合体の形成は、例えば、サイズ排除クロマトグラフィーを用い、ＣｐＧ　ＯＤＮの高分子量側へのシフトを２４０~２８０ｎｍ（例えば、２６０ｎｍ）における吸収をモニタリングすることによって確認することができる。 The preparation method of the complex of the present invention will be described using CpG-ODN and LNT complex as an example. LNT is dissolved in 0.05 to 2N, preferably 0.1 to 1.5N alkaline aqueous solution (for example, 0.25N sodium hydroxide aqueous solution) and left at 1 ° C to 40 ° C for 10 hours to 4 days (for example, Left overnight at room temperature) to prepare a single-stranded LNT aqueous solution (eg, 50 mg / ml LNT aqueous solution). The LNT aqueous solution and a separately prepared CpG aqueous solution (for example, 100 μM CpG aqueous solution) are mixed at a molar ratio (LNT / ODN) of 0.005 to 1.0, and then an acidic buffered aqueous solution (for example, NaH2PO4) is added to neutralize and maintained at 1-40 ° C. for 6 hours to 4 days (eg, overnight at 4 ° C.) to complete the complexation. In addition, you may add and mix LNT aqueous solution last for the said composite. Formation of the complex can be confirmed, for example, by using size exclusion chromatography and monitoring the absorption of CpG ODN toward the high molecular weight side at 240 to 280 nm (for example, 260 nm).
　一態様において、本発明の複合体は、竿状の粒子の形態を呈する。粒子径は、材料として用いるβ−１，３−グルカン（例、シゾフィラン）が天然で三重螺旋構造を呈することにより形成する粒子の径と同等であり、通常平均粒子径が１０−１００ｎｍ、好ましくは２０−５０ｎｍである。該粒子径は、複合体を水に溶解し、Ｍａｌｖｅｒｎ　Ｉｎｓｔｒｕｍｅｎｔｓ　Ｚｅｔａ　Ｓｉｚｅｒを用いて８０℃の条件で動的光散乱法により計測することができる。 In one aspect, the composite of the present invention takes the form of bowl-like particles. The particle diameter is the same as the diameter of a particle formed by β-1,3-glucan (eg, schizophyllan) used as a material naturally having a triple helical structure, and the average particle diameter is usually 10-100 nm, preferably 20-50 nm. The particle diameter can be measured by a dynamic light scattering method at 80 ° C. using a Malvern Instruments Zeta Sizer after dissolving the complex in water.
　本発明の複合体は、好ましくは単離されている。「単離された複合体」の純度（評価対象物の総重量に占める目的とする複合体重量の百分率）は、通常７０％以上、好ましくは８０％以上、より好ましくは９０％以上、更に好ましくは９９％以上である。 The complex of the present invention is preferably isolated. The purity of the “isolated complex” (percentage of the target complex weight in the total weight of the object to be evaluated) is usually 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably Is 99% or more.
　さらに、本発明の複合体は、抗がん活性のほか、優れた免疫賦活活性を有し、特に、Ｋ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、Ｂ細胞（好ましくは、ヒトＢ細胞）を活性化してＩＬ−６を産生させる活性）と、Ｄ型ＣｐＧ　ＯＤＮに特有の免疫賦活活性（例えば、形質細胞様樹状細胞（好ましくはヒト形質細胞様樹状細胞）を活性化してＩＦＮ−αを産生させる活性）の両方を有するので、免疫賦活剤等としても効果を付与し得るため有利でありうる。例えば、Ｋ型ＣｐＧ　ＯＤＮ（例えば、配列番号２、１１、１２とＳＰＧを含む複合体及びＫ型ＣｐＧ　ＯＤＮ（例えば、配列番号２）とＳＰＧを含む複合体（Ｋ３−ＳＰＧ）は、炎症応答誘導能（ｐａｎ−ＩＦＮ−ａ、ＩＬ−６等）、ウイルス接種個体における血清中抗原特異的ＩｇＧ抗体価（Ｔｏｔａｌ　ＩｇＧ，ＩｇＧ２ｃ等）の増強作用、ウイルス接種個体における抗原特異的サイトカイン産生能（ＩＦＮ−γ，ＩＬ２等）、ウイルスに対する感染防御効果を奏するものとしても有利でありうる。 Furthermore, the complex of the present invention has excellent immunostimulatory activity in addition to anticancer activity, and in particular, immunostimulatory activity peculiar to K-type CpG ODN (for example, B cells (preferably human B cells)) And the immunostimulatory activity peculiar to D-type CpG ODN (eg, plasmacytoid dendritic cells (preferably human plasmacytoid dendritic cells) are activated to activate IFN- Since it has both of (activity which produces (alpha)), since an effect can be provided also as an immunostimulant etc., it may be advantageous. For example, a K-type CpG ODN (for example, a complex containing SEQ ID NO: 2, 11, 12 and SPG and a complex containing a K-type CpG ODN (for example, SEQ ID NO: 2) and SPG (K3-SPG) induces an inflammatory response. Potency (pan-IFN-a, IL-6, etc.), serum antigen-specific IgG antibody titer (total IgG, IgG2c, etc.) in virus-inoculated individuals, antigen-specific cytokine production ability (IFN- (γ, IL2, etc.), which can also be effective as an anti-infection effect against viruses.
　（医薬組成物）
　本発明は、上記本発明のオリゴデオキシヌクレオチド又は上記本発明の複合体を含む、医薬組成物を提供するものである。本発明の医薬組成物は、上記本発明のオリゴデオキシヌクレオチド又は上記本発明の複合体を常套手段に従って製剤化することにより得ることができる。本発明の医薬組成物は、本発明のオリゴデオキシヌクレオチド又は複合体と薬理学的に許容され得る担体を含む。また、本医薬組成物は抗原を更に含んでいても良い。このような医薬組成物は、経口又は非経口投与に適する剤形として提供される。 (Pharmaceutical composition)
The present invention provides a pharmaceutical composition comprising the oligodeoxynucleotide of the present invention or the complex of the present invention. The pharmaceutical composition of the present invention can be obtained by formulating the oligodeoxynucleotide of the present invention or the complex of the present invention according to conventional means. The pharmaceutical composition of the present invention comprises the oligodeoxynucleotide or complex of the present invention and a pharmacologically acceptable carrier. The pharmaceutical composition may further contain an antigen. Such a pharmaceutical composition is provided as a dosage form suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明のオリゴデオキシヌクレオチド又は複合体を通常注射剤に用いられる無菌の水性溶媒に溶解又は懸濁することによって調製できる。注射用の水性溶媒としては、例えば、蒸留水；生理的食塩水；リン酸緩衝液、炭酸緩衝液、トリス緩衝液、酢酸緩衝液等の緩衝液等が使用できる。このような水性溶媒のｐＨは５~１０が挙げられ、好ましくは６~８である。調製された注射液は、適当なアンプルに充填されることが好ましい。 As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving or suspending the oligodeoxynucleotide or complex of the present invention in a sterile aqueous solvent usually used for injection. Examples of the aqueous solvent for injection include distilled water; physiological saline; phosphate buffer, carbonate buffer, Tris buffer, acetate buffer, and other buffer solutions. The pH of such an aqueous solvent is 5 to 10, preferably 6 to 8. The prepared injection solution is preferably filled in a suitable ampoule.
　また、本発明のオリゴデオキシヌクレオチド又は複合体の懸濁液を、真空乾燥、凍結乾燥等の処理に付すことにより、本発明のオリゴデオキシヌクレオチド又は複合体の粉末製剤を調製することもできる。本発明のオリゴデオキシヌクレオチド又は複合体を粉末状態で保存し、使用時に該粉末を注射用の水性溶媒で分散することにより、使用に供することができる。 In addition, a powder formulation of the oligodeoxynucleotide or complex of the present invention can also be prepared by subjecting the suspension of the oligodeoxynucleotide or complex of the present invention to a treatment such as vacuum drying or freeze drying. The oligodeoxynucleotide or complex of the present invention can be stored in a powder state, and can be used by dispersing the powder with an aqueous solvent for injection at the time of use.
　医薬組成物中の本発明のオリゴデオキシヌクレオチド又は複合体の含有量は、通常、医薬組成物全体の約０．１~１００重量％、好ましくは約１~９９重量％、さらに好ましくは約１０~９０重量％程度である。 The content of the oligodeoxynucleotide or complex of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by weight, preferably about 1 to 99% by weight, more preferably about 10 to 10% by weight of the whole pharmaceutical composition. About 90% by weight.
　本発明の医薬組成物は、有効成分として、本発明のオリゴデオキシヌクレオチド又は複合体を単独で含有していてもよく、本発明のオリゴデオキシヌクレオチド又は複合体を他の有効成分と組み合わせて含有していてもよい。 The pharmaceutical composition of the present invention may contain the oligodeoxynucleotide or complex of the present invention alone as an active ingredient, and contains the oligodeoxynucleotide or complex of the present invention in combination with other active ingredients. It may be.
　（医薬用途）
　本発明のオリゴデオキシヌクレオチド及び複合体は、単独で抗がん作用を有することが見出された。このような効果は、アジュバント剤として開発されてきた本発明の特性からは予想外の効果であるといえる。それゆえ、これまでのアジュバントとしての使用のされ方、すなわち、がん抗原とともに投与することを必要とすることなく、しかも、特定のがん種に限定されることなく、汎用的な抗がん剤として身体にマイルドに作用する抗がん剤が提供されることになる。また、免疫賦活活性ももちろん有することから、他の疾患に対する免役賦活活性も期待され、体力の弱ったがん患者に対して相乗的な効果を有することも期待される。 (Medical use)
The oligodeoxynucleotides and conjugates of the present invention have been found to have anticancer activity alone. Such an effect can be said to be an unexpected effect from the characteristics of the present invention that have been developed as an adjuvant. Therefore, it can be used as an adjuvant so far, that is, it is not necessary to administer with a cancer antigen, and it is not limited to a specific cancer type. An anticancer agent that acts mildly on the body as an agent will be provided. In addition, since it also has immunostimulatory activity, it can also be expected to have immune activation activity against other diseases, and to have a synergistic effect on cancer patients with weak physical strength.
　抗がん作用に加え、本発明は、優れた免疫賦活活性を有するので、本発明のオリゴデオキシヌクレオチド、複合体及び医薬組成物は、免疫賦活剤として用いることができる。本発明のオリゴデオキシヌクレオチド、複合体又は医薬組成物を哺乳動物（ヒト等の霊長類、マウス等のげっ歯類等）に投与することにより、該哺乳動物における免疫反応を惹起することができる。特に、本発明の複合体は、Ｄ型ＣｐＧ　ＯＤＮの活性特性を有し、末梢血単核球を刺激して、Ｉ型インターフェロン（Ｐａｎ−ＩＦＮ−α、ＩＦＮ−α２等）及びＩＩ型インターフェロン（ＩＦＮ−γ）の両方を大量に産生させるので、Ｉ型インターフェロン産生誘導剤、ＩＩ型インターフェロン産生誘導剤、Ｉ型及びＩＩ型インターフェロン産生誘導剤として有用である。Ｉ型及びＩＩ型インターフェロンの両方の産生を誘導することから、本発明の複合体及びこれを含有する医薬組成物は、Ｉ型及びＩＩ型インターフェロンのいずれか一方又は両方が有効な疾患の予防又は治療に有用である。 In addition to anti-cancer activity, the present invention has excellent immunostimulatory activity, so the oligodeoxynucleotide, complex and pharmaceutical composition of the present invention can be used as an immunostimulator. By administering the oligodeoxynucleotide, complex or pharmaceutical composition of the present invention to a mammal (a primate such as a human, a rodent such as a mouse), an immune reaction in the mammal can be induced. In particular, the complex of the present invention has the activity characteristics of D-type CpG ODN and stimulates peripheral blood mononuclear cells to give type I interferons (Pan-IFN-α, IFN-α2, etc.) and type II interferons ( Since both IFN-γ) are produced in large quantities, they are useful as type I interferon production inducers, type II interferon production inducers, type I and type II interferon production inducers. Since it induces the production of both type I and type II interferons, the complex of the present invention and the pharmaceutical composition containing the same prevent or prevent diseases in which one or both of type I and type II interferons are effective. Useful for treatment.
　医薬用途の実現方法としては、例えば（ａ）本発明のオリゴデオキシヌクレオチド、又は本発明の複合体を含む組成物を、癌の患者または癌に罹患する可能性のあるヒトに投与することにより、該投与を受けた対象中の細胞傷害性Ｔリンパ球（ＣＴＬ）を抗原特異的に活性化させ、直接的に（単剤効果として）癌を予防・治療することができる。 As a method for realizing the pharmaceutical use, for example, (a) by administering the oligodeoxynucleotide of the present invention or the composition containing the complex of the present invention to a cancer patient or a human who is likely to suffer from cancer, Cytotoxic T lymphocytes (CTL) in a subject who has received the administration can be antigen-specifically activated to prevent or treat cancer directly (as a single agent effect).
　本明細書において「被験体（者）」とは、本発明の診断または検出、あるいは治療等の対象となる対象（例えば、ヒト等の生物または生物から取り出した細胞、血液、血清等）をいう。 In the present specification, the “subject (person)” refers to a subject to be diagnosed or detected or treated according to the present invention (for example, an organism such as a human or a cell, blood, serum, etc. removed from the organism). .
　本明細書において「薬剤」、「剤」または「因子」（いずれも英語ではａｇｅｎｔに相当する）は、広義には、交換可能に使用され、意図する目的を達成することができる限りどのような物質または他の要素（例えば、光、放射能、熱、電気などのエネルギー）でもあってもよい。そのような物質としては、例えば、タンパク質、ポリペプチド、オリゴペプチド、ペプチド、ポリヌクレオチド、オリゴヌクレオチド、ヌクレオチド、核酸（例えば、ｃＤＮＡ、ゲノムＤＮＡのようなＤＮＡ、ｍＲＮＡのようなＲＮＡを含む）、ポリサッカリド、オリゴサッカリド、脂質、有機低分子（例えば、ホルモン、リガンド、情報伝達物質、有機低分子、コンビナトリアルケミストリで合成された分子、医薬品として利用され得る低分子（例えば、低分子リガンドなど）など）、これらの複合分子が挙げられるがそれらに限定されない。ポリヌクレオチドに対して特異的な因子としては、代表的には、そのポリヌクレオチドの配列に対して一定の配列相同性を（例えば、７０％以上の配列同一性）もって相補性を有するポリヌクレオチド、プロモーター領域に結合する転写因子のようなポリペプチドなどが挙げられるがそれらに限定されない。ポリペプチドに対して特異的な因子としては、代表的には、そのポリペプチドに対して特異的に指向された抗体またはその誘導体あるいはその類似物（例えば、単鎖抗体）、そのポリペプチドがレセプターまたはリガンドである場合の特異的なリガンドまたはレセプター、そのポリペプチドが酵素である場合、その基質などが挙げられるがそれらに限定されない。 In the present specification, “drug”, “agent” or “factor” (both corresponding to “agent” in English) are used interchangeably in a broad sense, and so long as they can achieve their intended purpose. It may also be a substance or other element (eg energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNA such as cDNA, genomic DNA, RNA such as mRNA), poly Saccharides, oligosaccharides, lipids, small organic molecules (for example, hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (for example, small molecule ligands, etc.)) , These complex molecules are included, but not limited thereto. As a factor specific for a polynucleotide, typically, a polynucleotide having a certain sequence homology to the sequence of the polynucleotide (for example, 70% or more sequence identity) and complementarity, Examples include, but are not limited to, a polypeptide such as a transcription factor that binds to the promoter region. Factors specific for a polypeptide typically include an antibody specifically directed against the polypeptide or a derivative or analog thereof (eg, a single chain antibody), and the polypeptide is a receptor. Alternatively, specific ligands or receptors in the case of ligands, and substrates thereof when the polypeptide is an enzyme include, but are not limited to.
　本明細書において「治療」とは、ある疾患または障害（例えば、がん、アレルギー）について、そのような状態になった場合に、そのような疾患または障害の悪化を防止、好ましくは、現状維持、より好ましくは、軽減、さらに好ましくは消退させることをいい、患者の疾患、もしくは疾患に伴う１つ以上の症状の、症状改善効果あるいは予防効果を発揮しうることを含む。事前に診断を行って適切な治療を行うことは「コンパニオン治療」といい、そのための診断薬を「コンパニオン診断薬」ということがある。 As used herein, “treatment” refers to prevention of worsening of a disease or disorder when a disease or disorder (eg, cancer, allergy) occurs, preferably, maintaining the status quo. More preferably, it means reduction, further preferably elimination, which includes the ability to exert a symptom improving effect or a preventive effect on one or more symptoms associated with a patient's disease or disease. Diagnosing in advance and performing appropriate treatment is referred to as “companion treatment”, and the diagnostic agent therefor is sometimes referred to as “companion diagnostic agent”.
　本明細書において「治療薬（剤）」とは、広義には、目的の状態（例えば、がん、アレルギー等の疾患など）を治療できるあらゆる薬剤をいう。本発明の一実施形態において「治療薬」は、有効成分と、薬理学的に許容される１つもしくはそれ以上の担体とを含む医薬組成物であってもよい。医薬組成物は、例えば有効成分と上記担体とを混合し、製剤学の技術分野において知られる任意の方法により製造できる。また治療薬は、治療のために用いられる物であれば使用形態は限定されず、有効成分単独であってもよいし、有効成分と任意の成分との混合物であってもよい。また上記担体の形状は特に限定されず、例えば、固体または液体（例えば、緩衝液）であってもよい。なおがん、アレルギー等の治療薬は、がん、アレルギー等の予防のために用いられる薬物（予防薬）、またはがん、アレルギー等の抑制剤を含む。 In this specification, the term “therapeutic agent (agent)” refers to any drug that can treat a target condition (for example, diseases such as cancer, allergy, etc.) in a broad sense. In one embodiment of the present invention, the “therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced by any method known in the technical field of pharmaceutics, for example, by mixing the active ingredient and the carrier. In addition, the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be an active ingredient alone or a mixture of an active ingredient and an arbitrary ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution). The therapeutic agent for cancer, allergy and the like includes a drug (preventive agent) used for preventing cancer, allergy and the like, or a suppressor for cancer, allergy and the like.
　本明細書において「予防」とは、ある疾患または障害（例えば、アレルギー）について、そのような状態になる前に、そのような状態にならないようにすることをいう。本発明の薬剤を用いて、診断を行い、必要に応じて本発明の薬剤を用いて例えば、アレルギー等の予防をするか、あるいは予防のための対策を講じることができる。 In the present specification, “prevention” means that a certain disease or disorder (for example, allergy) is prevented from becoming such a state before it becomes such a state. Diagnosis can be performed using the drug of the present invention, and for example, allergies can be prevented using the drug of the present invention, or countermeasures for prevention can be taken as necessary.
　本明細書において「予防薬（剤）」とは、広義には、目的の状態（例えば、アレルギー等の疾患など）を予防できるあらゆる薬剤をいう。 In this specification, the term “prophylactic agent (agent)” refers to any agent that can prevent a target condition (for example, a disease such as allergy) in a broad sense.
　本明細書において「キット」とは、通常２つ以上の区画に分けて、提供されるべき部分（例えば、検査薬、診断薬、治療薬、抗体、標識、説明書など）が提供されるユニットをいう。安定性等のため、混合されて提供されるべきでなく、使用直前に混合して使用することが好ましいような組成物の提供を目的とするときに、このキットの形態は好ましい。そのようなキットは、好ましくは、提供される部分（例えば、検査薬、診断薬、治療薬をどのように使用するか、あるいは、試薬をどのように処理すべきかを記載する指示書または説明書を備えていることが有利である。本明細書においてキットが試薬キットとして使用される場合、キットには、通常、検査薬、診断薬、治療薬、抗体等の使い方などを記載した指示書などが含まれる。 In the present specification, the “kit” is a unit provided with a portion to be provided (eg, a test agent, a diagnostic agent, a therapeutic agent, an antibody, a label, an instruction, etc.) usually divided into two or more compartments. Say. This kit form is preferred when it is intended to provide a composition that should not be provided in admixture for stability or the like, but preferably used in admixture immediately before use. Such kits preferably include instructions or instructions that describe how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how the reagents should be processed). In the present specification, when the kit is used as a reagent kit, the kit usually contains instructions including usage of test agents, diagnostic agents, therapeutic agents, antibodies, etc. Is included.
　本明細書において「指示書」は、本発明を使用する方法を医師または他の使用者に対する説明を記載したものである。この指示書は、本発明の検出方法、診断薬の使い方、または医薬などを投与することを指示する文言が記載されている。また、指示書には、投与部位として、経口、食道への投与（例えば、注射などによる）することを指示する文言が記載されていてもよい。この指示書は、本発明が実施される国の監督官庁（例えば、日本であれば厚生労働省、米国であれば食品医薬品局（ＦＤＡ）など）が規定した様式に従って作成され、その監督官庁により承認を受けた旨が明記される。指示書は、いわゆる添付文書（ｐａｃｋａｇｅ　ｉｎｓｅｒｔ）であり、通常は紙媒体で提供されるが、それに限定されず、例えば、電子媒体（例えば、インターネットで提供されるホームページ、電子メール）のような形態でも提供され得る。 In the present specification, the “instruction sheet” describes the method for using the present invention for a doctor or other user. This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given. In addition, the instructions may include a word indicating that the administration site is oral or esophageal administration (for example, by injection). This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction sheet is a so-called package insert and is usually provided as a paper medium, but is not limited thereto, and is in the form of, for example, an electronic medium (for example, a home page or e-mail provided on the Internet). But it can be provided.
　（好ましい実施形態の態様）
　以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。また、本発明の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。 (Aspects of preferred embodiments)
Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification. It will also be appreciated that the following embodiments of the invention may be used alone or in combination.
　＜単剤形態＞
　１つの局面では、本発明は、（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、（ｂ）β—１，３−グルカンとを含む、複合体を含む抗がん剤を提供する。本発明では、本発明の複合体自体が抗がん剤として作用することを見出した。従来は、この複合体は、アジュバントとして用いられることを本発明者らが見出し出願したにすぎず、直接、単剤として抗がん剤として用いることができるとは予想していなかった。したがって、がん抗原なしで使用されるという点で予想外の効果をもたらすといえる。 <Single agent form>
In one aspect, the invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. Provided is an anticancer agent containing a complex comprising oligodeoxynucleotides arranged on the 3 ′ side and (b) β-1,3-glucan. In the present invention, it was found that the complex itself of the present invention acts as an anticancer agent. Conventionally, the present inventors have only found out and applied for the use of this complex as an adjuvant, and have not anticipated that it can be directly used as an anticancer agent as a single agent. Therefore, it can be said that it produces an unexpected effect in that it is used without a cancer antigen.
　１つの実施形態では、本発明の抗がん剤は、がん抗原なしで投与されることを特徴とする。 In one embodiment, the anticancer agent of the present invention is administered without a cancer antigen.
　別の実施形態では、本発明の抗がん剤は、細網内皮系および／またはリンパ節に送達されるように投与されることを特徴とする。好ましくは、前記細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む。例示的には、前記細網内皮系は脾臓および／または肝臓を含む。したがって、本発明の抗がん剤は、腫瘍および貪食細胞を含む細網内皮系臓器（脾臓、肝臓など）および／またはリンパ節に送達されるように投与されることを特徴とする。理論に束縛されることを望まないが、本発明の複合体は、腫瘍および貪食細胞に送達され、そこで、がんの死細胞を細網内皮系臓器（脾臓、肝臓など）にリクルートすることが示された。これにより、身体内のがん細胞をさらに駆逐することができると考えられる。したがって、本発明は、アジュバントとして特定のがん抗原を用いて特定のがんに対するのではなく、身体内に存在する任意のがん細胞を殺傷し得るということができ、従来にない抗がん剤を提供することができる、という点で顕著な効果をもたらすといえる。 In another embodiment, the anticancer agent of the present invention is administered so as to be delivered to the reticuloendothelial system and / or lymph nodes. Preferably, the reticuloendothelial system and / or lymph nodes comprise tumors and phagocytic cells. Illustratively, the reticuloendothelial system includes the spleen and / or liver. Therefore, the anticancer agent of the present invention is characterized in that it is administered so as to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes including tumors and phagocytic cells. Without wishing to be bound by theory, the complexes of the invention can be delivered to tumors and phagocytic cells, where cancer dead cells can be recruited to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.
　したがって、より好ましい実施形態では、本発明の前記抗がん剤は、がん抗原なしで、腫瘍および貪食細胞に送達されるように投与されることを特徴とする。 Therefore, in a more preferred embodiment, the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.
　このような送達手法は、任意の手法を用いることができ、例えば、前記投与は全身性投与を挙げることができるがこれに限定されない。好ましくは全身性投与であり、全身性投与としては、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与等を挙げることができる。 Such a delivery method may be any method, and for example, the administration may include systemic administration, but is not limited thereto. Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.
　１つの実施形態では、本発明で用いられるオリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）などを挙げることができるがこれらに限定されない。 In one embodiment, the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6), K3-dA35 (SEQ ID NO: 7), and the like.
　１つの実施形態では、本発明で用いられるβ—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホラン、ラミナラン等であってもよい。 In one embodiment, β-1,3-glucan used in the present invention may be schizophyllan (SPG), lentinan, scleroglucan, curdlan, perchiman, glyforan, laminaran and the like.
　好ましい実施形態では、本発明の複合体は、Ｋ３−ＳＰＧまたはその類似体である。ここで、類自体とは、ＣｐＧ側で構造がＫ３に類似するもの、βグルカン側で構造がＳＰＧに類似するもの等を挙げることができるがこれらに限定されない。 In a preferred embodiment, the complex of the present invention is K3-SPG or an analogue thereof. Examples of the class itself include, but are not limited to, those having a structure similar to K3 on the CpG side and those having a structure similar to SPG on the β-glucan side.
　なお、抗がん作用は、種々の機構によることから、がんの死細胞を脾臓に集積させるため等の用途は容易に思いつくものではない。特に、全身性投与において、腫瘍に集積し、死んだ腫瘍細胞を脾臓などの組織に集積させるための、用途は思いつくものではない。また、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）αの発現またはその促進効果もまた、抗がん作用とは異なる機構によるものであり、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）αの発現またはその促進は抗がん以外でも発揮し得ることから、相互に容易に思いつくものではない。したがって、本発明のＣｐＧ−βグルカン複合体の各用途（抗がん用途（単剤での）、がんの死細胞を脾臓に集積させる用途、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）αの発現またはその促進のための用途）は、相互に容易に想到し得たとは言えない関係にあるといえる。 In addition, since the anti-cancer action is due to various mechanisms, it is not easy to conceive uses such as collecting dead cancer cells in the spleen. In particular, in systemic administration, the use for accumulating in a tumor and accumulating dead tumor cells in tissues such as the spleen is not conceivable. In addition, the expression of interleukin 12 (IL12) and / or interferon (IFN) α or its promoting effect is also due to a mechanism different from the anticancer action, and interleukin 12 (IL12) and / or interferon (IFN) ) Since the expression of α or its promotion can be exerted other than anti-cancer, it is not easy to come up with each other. Therefore, each use of the CpG-β glucan complex of the present invention (anti-cancer use (single agent), use to accumulate cancer dead cells in the spleen, interleukin 12 (IL12) and / or interferon (IFN)) It can be said that there is a relationship that cannot be easily conceived with each other.
　＜細網内皮系（脾臓および／または肝臓を含む）および／またはリンパ節集積剤＞
　別の局面において、本発明は、（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、（ｂ）β—１，３−グルカンとを含む、複合体を含む、がんの死細胞を細網内皮系（脾臓および／または肝臓を含む）および／またはリンパ節に集積させるための組成物を提供する。理論に束縛されることを望まないが、本発明の複合体は、がんの死細胞を細網内皮系（脾臓および／または肝臓を含む）および／またはリンパ節に集積させることができることを見出した。実施例で例証されているように、Ｋ３−ＳＰＧのような本発明の複合体による処置は、ＩＬ１２ｐ４０およびＩＦＮ−Ｉの両方に依存する態様で、腫瘍細胞死を誘発することが実証された。このような作用を複合体が有することは従来予想されておらず、その意味で予想外の作用効果が達成されたといえる。すなわち、ＣｐＧが腫瘍微小環境における食細胞に標的化されるというものである。がんの死細胞が細網内皮系（脾臓および／または肝臓を含む）および／またはリンパ節に集積されると、その後、放出された腫瘍死細胞は複数の腫瘍抗原に対する抗腫瘍ＣＴＬを誘発し、身体にあるがん細胞が散弾銃で攻撃されるかのように殺傷され、根治させることも可能である。理論に束縛されることを望まないが、腫瘍微小環境におけるＩＬ１２およびＩＦＮ−Ｉサイトカインの両方を産生することは、Ｋ３−ＳＰＧ単剤治療に必須とまではいえないが、重要である。 <Reticuloendothelial system (including spleen and / or liver) and / or lymph node accumulation agent>
In another aspect, the present invention provides an oligodeoxynucleotide comprising (a) a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. 3'-side oligodeoxynucleotide and (b) β-1,3-glucan-containing complex, cancerous dead cells containing reticuloendothelial system (spleen and / or liver) And / or a composition for accumulation in lymph nodes. While not wishing to be bound by theory, it has been found that the complex of the present invention can accumulate cancer dead cells in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes. It was. As illustrated in the Examples, treatment with a complex of the invention such as K3-SPG has been demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I. It has not been conventionally expected that the complex has such an action, and in that sense, it can be said that an unexpected action and effect has been achieved. That is, CpG is targeted to phagocytic cells in the tumor microenvironment. Once cancer dead cells accumulate in the reticuloendothelial system (including spleen and / or liver) and / or lymph nodes, the released tumor dead cells then induce anti-tumor CTL against multiple tumor antigens. The cancer cells in the body can be killed and cured as if they were attacked with a shotgun. Without wishing to be bound by theory, it is important, although not necessarily essential for K3-SPG monotherapy, to produce both IL12 and IFN-I cytokines in the tumor microenvironment.
　１つの実施形態では、本発明で用いられるオリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）からなる群より選択される。 In one embodiment, the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
　別の実施形態では、本発明で用いられるβ—１，３−グルカンはシゾフィラン（ＳＰＧ）、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される。 In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, parkan, glyphoran and laminaran.
　好ましい実施形態では、本発明の複合体は、Ｋ３−ＳＰＧである。 In a preferred embodiment, the complex of the present invention is K3-SPG.
　１つの実施形態では、本発明の組成物が対象とする細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む。例示的には、前記細網内皮系は脾臓および／または肝臓を含む。したがって、本発明の組成物は、腫瘍および貪食細胞を含む細網内皮系臓器（脾臓、肝臓など）および／またはリンパ節に送達されるように投与されることを特徴とする。理論に束縛されることを望まないが、本発明の複合体は、腫瘍および貪食細胞に送達され、そこで、がんの死細胞を細網内皮系臓器（脾臓、肝臓など）にリクルートすることが示された。これにより、身体内のがん細胞をさらに駆逐することができると考えられる。したがって、本発明は、アジュバントとして特定のがん抗原を用いて特定のがんに対するのではなく、身体内に存在する任意のがん細胞を殺傷し得るということができ、従来にない抗がん剤を提供することができる、という点で顕著な効果をもたらすといえる。 In one embodiment, the reticuloendothelial system and / or lymph nodes targeted by the composition of the present invention comprises tumors and phagocytic cells. Illustratively, the reticuloendothelial system includes the spleen and / or liver. Accordingly, the composition of the present invention is characterized in that it is administered so as to be delivered to reticuloendothelial organs (spleen, liver, etc.) and / or lymph nodes containing tumors and phagocytic cells. Without wishing to be bound by theory, the complexes of the invention can be delivered to tumors and phagocytic cells, where cancer dead cells can be recruited to reticuloendothelial organs (spleen, liver, etc.). Indicated. Thereby, it is considered that cancer cells in the body can be further destroyed. Therefore, it can be said that the present invention can kill any cancer cell present in the body, not against a specific cancer using a specific cancer antigen as an adjuvant, It can be said that a remarkable effect is brought about in that an agent can be provided.
　したがって、より好ましい実施形態では、本発明の前記抗がん剤は、がん抗原なしで、腫瘍および貪食細胞に送達されるように投与されることを特徴とする。 Therefore, in a more preferred embodiment, the anticancer agent of the present invention is administered so as to be delivered to tumors and phagocytic cells without a cancer antigen.
　このような送達手法は、任意の手法を用いることができ、例えば、前記投与は全身性投与を挙げることができるがこれに限定されない。好ましくは全身性投与であり、全身性投与としては、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与等を挙げることができる。 Such a delivery method may be any method, and for example, the administration may include systemic administration, but is not limited thereto. Systemic administration is preferred, and examples of systemic administration include intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, and intramuscular administration.
　＜ＩＬ１２および／またはＩＦＮ発現促進剤＞
　さらに別の局面では、本発明は、（ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、（ｂ）β—１，３−グルカンとを含む、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）γの発現またはその促進のための組成物を提供する。腫瘍微小環境におけるＩＬ１２およびＩＦＮ−Ｉサイトカインの両方を産生することは、Ｋ３−ＳＰＧ単剤治療における重要な作用効果であり、このような効果は、抗がん剤としての作用のほか、他の用途においても重要である。そのような処置の対象としては、がんのほか、ウイルスなどの慢性感染症疾患、ウイルス感染予防などを挙げることができるがそれらに限定されない。 <IL12 and / or IFN expression promoter>
In yet another aspect, the present invention provides (a) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is a humanized K-type CpG oligodeoxynucleotide. For expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, which includes an oligodeoxynucleotide and (b) β-1,3-glucan arranged on the 3 ′ side of A composition is provided. Producing both IL12 and IFN-I cytokines in the tumor microenvironment is an important effect in K3-SPG monotherapy, such as acting as an anti-cancer agent and other It is also important in applications. Examples of such treatment include, but are not limited to, cancer, chronic infectious diseases such as viruses, and prevention of viral infection.
　１つの実施形態では、本発明で用いられるオリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）からなる群より選択される。 In one embodiment, the oligodeoxynucleotide used in the present invention is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4). , K3-dA25 (SEQ ID NO: 5), K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
　別の実施形態では、本発明で用いられるβ—１，３−グルカンはシゾフィラン（ＳＰＧ）、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される。 In another embodiment, the β-1,3-glucan used in the present invention is selected from the group consisting of schizophyllan (SPG), scleroglucan, curdlan, parkan, glyphoran and laminaran.
　好ましい実施形態では、本発明の複合体は、Ｋ３−ＳＰＧである。 In a preferred embodiment, the complex of the present invention is K3-SPG.
　（医薬品、剤型等）
　本発明は、上記種々の形態の医薬（治療薬または予防薬）として提供される。 (Pharmaceuticals, dosage forms, etc.)
The present invention is provided as the above-mentioned various forms of medicaments (therapeutic or preventive).
　治療薬の投与経路は、治療に際して効果的なものを使用するのが好ましく、例えば、静脈内、皮下、筋肉内、腹腔内、または経口投与等であってもよい。投与形態としては、例えば、注射剤、カプセル剤、錠剤、顆粒剤等であってもよい。本発明の成分を投与する場合には、注射剤として用いることが効果的である。注射用の水溶液は、例えば、バイアル、またはステンレス容器で保存してもよい。また注射用の水溶液は、例えば生理食塩水、糖（例えばトレハロース）、ＮａＣｌ、またはＮａＯＨ等を配合してもよい。また治療薬は、例えば、緩衝剤（例えばリン酸塩緩衝液）、安定剤等を配合してもよい。 The administration route of the therapeutic agent is preferably one that is effective in the treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, capsule, tablet, granule or the like. When administering the component of this invention, using as an injection is effective. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. The aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, or NaOH. The therapeutic agent may contain, for example, a buffer (for example, phosphate buffer), a stabilizer and the like.
　一般的に、本発明の組成物、医薬、治療剤、予防剤等は、治療有効量の治療剤または有効成分、および薬学的に許容しうるキャリアもしくは賦形剤を含む。本明細書において「薬学的に許容しうる」は、動物、そしてより詳細にはヒトにおける使用のため、政府の監督官庁に認可されたか、あるいは薬局方または他の一般的に認められる薬局方に列挙されていることを意味する。本明細書において使用される「キャリア」は、治療剤を一緒に投与する、希釈剤、アジュバント、賦形剤、またはビヒクルを指す。このようなキャリアは、無菌液体、例えば水および油であることも可能であり、石油、動物、植物または合成起源のものが含まれ、限定されるわけではないが、ピーナツ油、ダイズ油、ミネラルオイル、ゴマ油等が含まれる。医薬を経口投与する場合は、水が好ましいキャリアである。医薬組成物を静脈内投与する場合は、生理食塩水および水性デキストロースが好ましいキャリアである。好ましくは、生理食塩水溶液、並びに水性デキストロースおよびグリセロール溶液が、注射可能溶液の液体キャリアとして使用される。適切な賦形剤には、軽質無水ケイ酸、結晶セルロース、マンニトール、デンプン、グルコース、ラクトース、スクロース、ゼラチン、モルト、米、小麦粉、チョーク、シリカゲル、ステアリン酸ナトリウム、モノステアリン酸グリセロール、タルク、塩化ナトリウム、脱脂粉乳、グリセロール、プロピレン、グリコール、水、エタノール、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油６０、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩等が含まれる。組成物は、望ましい場合、少量の湿潤剤または乳化剤、あるいはｐＨ緩衝剤もまた含有することも可能である。これらの組成物は、溶液、懸濁物、エマルジョン、錠剤、ピル、カプセル、粉末、持続放出配合物等の形を取ることも可能である。伝統的な結合剤およびキャリア、例えばトリグリセリドを用いて、組成物を座薬として配合することも可能である。経口配合物は、医薬等級のマンニトール、ラクトース、デンプン、ステアリン酸マグネシウム、サッカリン・ナトリウム、セルロース、炭酸マグネシウムなどの標準的キャリアを含むことも可能である。適切なキャリアの例は、Ｅ．Ｗ．Ｍａｒｔｉｎ，Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅｓ（Ｍａｒｋ　Ｐｕｂｌｉｓｈｉｎｇ　Ｃｏｍｐａｎｙ，Ｅａｓｔｏｎ，Ｕ．Ｓ．Ａ）に記載される。このような組成物は、患者に適切に投与する形を提供するように、適切な量のキャリアと一緒に、治療有効量の療法剤、好ましくは精製型のものを含有する。配合物は、投与様式に適していなければならない。これらのほか、例えば、界面活性剤、賦形剤、着色料、着香料、保存料、安定剤、緩衝剤、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性促進剤、矯味剤等を含んでいてもよい。 Generally, the compositions, medicaments, therapeutic agents, prophylactic agents, etc. of the present invention comprise a therapeutically effective amount of a therapeutic agent or active ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable” refers to a licensed or otherwise recognized pharmacopoeia of a government for use in animals, and more particularly in humans, by a government supervisory authority. It means that it is enumerated. As used herein, “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such carriers can be sterile liquids, such as water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, minerals Oil, sesame oil, etc. are included. Water is a preferred carrier when the drug is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Preferably, saline solutions and aqueous dextrose and glycerol solutions are used as liquid carriers for injectable solutions. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, chloride Sodium, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hardening Castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts and the like are included. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. It is also possible to formulate the composition as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations may also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. Examples of suitable carriers are E.I. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, USA). Such compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation must be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffering agents, suspending agents, tonicity agents, binders, disintegrating agents, lubricants, fluidity Accelerators, flavoring agents and the like may be included.
　本発明の一実施形態において「塩」は、例えば、任意の酸性（例えばカルボキシル）基で形成されるアニオン塩、または任意の塩基性（例えばアミノ）基で形成されるカチオン塩を含む。塩類には無機塩または有機塩を含み、例えば、Ｂｅｒｇｅ　ｅｔ　ａｌ．，Ｊ．Ｐｈａｒｍ．Ｓｃｉ．，１９７７，６６，１−１９に記載されている塩が含まれる。また例えば、金属塩、アンモニウム塩、有機塩基との塩、無機酸との塩、有機酸との塩等が挙げられる。本発明の一実施形態において「溶媒和物」は、溶質および溶媒によって形成される化合物である。溶媒和物については例えば、Ｊ．Ｈｏｎｉｇｅｔ　ａｌ．，Ｔｈｅ　Ｖａｎ　Ｎｏｓｔｒａｎｄ　Ｃｈｅｍｉｓｔ’ｓ　Ｄｉｃｔｉｏｎａｒｙ　Ｐ６５０　（１９５３）を参照できる。溶媒が水であれば形成される溶媒和物は水和物である。この溶媒は、溶質の生物活性を妨げないものが好ましい。そのような好ましい溶媒の例として、特に限定するものではないが、水、または各種バッファーが挙げられる。本発明の一実施形態において「化学修飾」は、例えば、ＰＥＧもしくはその誘導体による修飾、フルオレセイン修飾、またはビオチン修飾等が挙げられる。 In one embodiment of the present invention, the “salt” includes, for example, an anion salt formed with any acidic (eg, carboxyl) group or a cation salt formed with any basic (eg, amino) group. Salts include inorganic salts or organic salts, for example, Berge et al. , J .; Pharm. Sci. , 1977, 66, 1-19. Examples thereof include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, and the like. In one embodiment of the present invention, a “solvate” is a compound formed by a solute and a solvent. For solvates, see, for example, J. Org. Honiget al. , The Van Nostrand Chemist's Dictionary P650 (1953). If the solvent is water, the solvate formed is a hydrate. This solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers. In one embodiment of the present invention, “chemical modification” includes, for example, modification with PEG or a derivative thereof, fluorescein modification, biotin modification, or the like.
　本発明を医薬として投与する場合、種々の送達（デリバリー）系が知られ、そしてこのような系を用いて、本発明の治療剤を適切な部位（例えば、食道）に投与することも可能であり、このような系には、例えばリポソーム、微小粒子、および微小カプセル中の被包：治療剤（例えば、ポリペプチド）を発現可能な組換え細胞の使用、受容体が仲介するエンドサイトーシスの使用；レトロウイルスベクターまたは他のベクターの一部としての療法核酸の構築などがある。導入法には、限定されるわけではないが、皮内、筋内、腹腔内、静脈内、皮下、鼻内、硬膜外、および経口経路が含まれる。好適な経路いずれによって、例えば注入によって、ボーラス（ｂｏｌｕｓ）注射によって、上皮または皮膚粘膜裏打ち（例えば口腔、直腸および腸粘膜など）を通じた吸収によって、医薬を投与することも可能であるし、必要に応じてエアロゾル化剤を用いて吸入器または噴霧器を使用しうるし、そして他の生物学的活性剤と一緒に投与することも可能である。投与は全身性または局所であることも可能である。本発明ががんに使用される場合、さらに、がん（病変部）に直接注入する等、適切な経路いずれかによって投与されうる。 When the present invention is administered as a pharmaceutical, various delivery systems are known, and such systems can be used to administer the therapeutic agent of the present invention to an appropriate site (eg, esophagus). Such systems include, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of recombinant cells capable of expressing therapeutic agents (eg, polypeptides), receptor-mediated endocytosis Use; such as the construction of therapeutic nucleic acids as part of a retroviral vector or other vector. Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. It is possible and necessary to administer the medicament by any suitable route, for example by infusion, by bolus injection, by absorption through epithelial or dermal mucosal lining (eg oral, rectal and intestinal mucosa, etc.) Accordingly, an inhaler or nebulizer can be used with an aerosolizing agent and can be administered with other biologically active agents. Administration can be systemic or local. When the present invention is used for cancer, it can be administered by any appropriate route such as direct injection into cancer (lesion).
　好ましい実施形態において、公知の方法に従って、ヒトへの投与に適応させた医薬組成物として、組成物を配合することができる。このような組成物は注射により投与することができる。代表的には、注射投与のための組成物は、無菌等張水性緩衝剤中の溶液である。必要な場合、組成物はまた、可溶化剤および注射部位での疼痛を和らげるリドカインなどの局所麻酔剤も含むことも可能である。一般的に、成分を別個に供給するか、または単位投薬型中で一緒に混合して供給し、例えば活性剤の量を示すアンプルまたはサシェなどの密封容器中、凍結乾燥粉末または水不含濃縮物として供給することができる。組成物を注入によって投与しようとする場合、無菌薬剤等級の水または生理食塩水を含有する注入ビンを用いて、分配することも可能である。組成物を注射によって投与しようとする場合、投与前に、成分を混合可能であるように、注射用の無菌水または生理食塩水のアンプルを提供することも可能である。 In a preferred embodiment, the composition can be formulated as a pharmaceutical composition adapted for human administration according to known methods. Such compositions can be administered by injection. Typically, compositions for injection administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. In general, the ingredients are supplied separately or mixed together in a unit dosage form, for example in a sealed container such as an ampoule or sachet indicating the amount of active agent, lyophilized powder or water-free concentration Can be supplied as a product. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
　本発明の組成物、医薬、治療剤、予防剤を中性型または塩型あるいは他のプロドラッグ（例えば、エステル等）で配合することも可能である。薬学的に許容しうる塩には、塩酸、リン酸、酢酸、シュウ酸、酒石酸などに由来する遊離型のカルボキシル基とともに形成されるもの、イソプロピルアミン、トリエチルアミン、２−エチルアミノエタノール、ヒスチジン、プロカインなどに由来するものなどの遊離型のアミン基とともに形成されるもの、並びにナトリウム、カリウム、アンモニウム、カルシウム、および水酸化第二鉄などに由来するものが含まれる。 It is also possible to mix the composition, medicament, therapeutic agent, and preventive agent of the present invention in a neutral form, salt form, or other prodrug (for example, ester). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine And those formed with free amine groups such as those derived from, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, and the like.
　特定の障害または状態の治療に有効な本発明の治療剤の量は、障害または状態の性質によって変動しうるが、当業者は本明細書の記載に基づき標準的臨床技術によって決定可能である。さらに、場合によって、ｉｎ　ｖｉｔｒｏアッセイを使用して、最適投薬量範囲を同定するのを補助することも可能である。配合物に使用しようとする正確な用量はまた、投与経路、および疾患または障害の重大性によっても変動しうるため、担当医の判断および各患者の状況に従って、決定すべきである。しかし、投与量は特に限定されないが、例えば、１回あたり０．００１、１、５、１０、１５、１００、または１０００ｍｇ／ｋｇ体重であってもよく、それらいずれか２つの値の範囲内であってもよい。投与間隔は特に限定されないが、例えば、１、７、１４、２１、または２８日あたりに１または２回投与してもよく、それらいずれか２つの値の範囲あたりに１または２回投与してもよい。投与量、投与間隔、投与方法は、患者の年齢や体重、症状、対象臓器等により、適宜選択してもよい。また治療薬は、治療有効量、または所望の作用を発揮する有効量の有効成分を含むことが好ましい。悪性腫瘍マーカーが、投与後に有意に減少した場合に、治療効果があったと判断してもよい。有効用量は、ｉｎ　ｖｉｔｒｏまたは動物モデル試験系から得られる用量−反応曲線から推定可能である。 The amount of the therapeutic agent of the invention effective for the treatment of a particular disorder or condition can vary depending on the nature of the disorder or condition, but can be determined by those skilled in the art by standard clinical techniques based on the description herein. Furthermore, in some cases, in vitro assays can be used to help identify optimal dosage ranges. The exact dose to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, and within the range of any two of these values There may be. The dosing interval is not particularly limited. For example, it may be administered once or twice per 1, 7, 14, 21, or 28 days, or once or twice per any two of these ranges. Also good. The dose, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target organ, etc. of the patient. The therapeutic agent preferably contains a therapeutically effective amount or an effective amount of an active ingredient that exhibits a desired action. If the malignant tumor marker is significantly decreased after administration, it may be determined that there is a therapeutic effect. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
　本発明の一実施形態において「患者」または「被験体」は、ヒト、またはヒトを除く哺乳動物（例えば、マウス、モルモット、ハムスター、ラット、ネズミ、ウサギ、ブタ、ヒツジ、ヤギ、ウシ、ウマ、ネコ、イヌ、マーモセット、サル、またはチンパンジー等の１種以上）を含む。 In one embodiment of the present invention, a “patient” or “subject” is a human or non-human mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, One or more of a cat, dog, marmoset, monkey, or chimpanzee).
　本発明の医薬組成物または治療剤もしくは予防剤はキットとして提供することができる。 The pharmaceutical composition or therapeutic agent or prophylactic agent of the present invention can be provided as a kit.
　特定の実施形態では、本発明は、本発明の組成物または医薬の１以上の成分が充填された、１以上の容器を含む、薬剤パックまたはキットを提供する。場合によって、このような容器に付随して、医薬または生物学的製品の製造、使用または販売を規制する政府機関によって規定された形で、政府機関による、ヒト投与のための製造、使用または販売の認可を示す情報を示すことも可能である。 In certain embodiments, the present invention provides a drug pack or kit comprising one or more containers filled with one or more components of the composition or medicament of the present invention. In some cases, associated with such containers, manufactured, used or sold for human administration by a government agency in a manner prescribed by the government agency that regulates the manufacture, use or sale of a pharmaceutical or biological product. It is also possible to indicate information indicating authorization.
　特定の実施形態において、本発明の成分を含む医薬組成物を、リポソーム、微小粒子、または微小カプセルを介して投与することができる。本発明の多様な態様において、このような組成物を用いて、本発明の成分の持続放出を達成することが有用である可能性もある。 In certain embodiments, a pharmaceutical composition comprising an ingredient of the present invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to achieve sustained release of the components of the present invention using such compositions.
　本発明の治療薬、予防薬等の医薬等としての製剤化手順は、当該分野において公知であり、例えば、日本薬局方、米国薬局方、他の国の薬局方などに記載されている。従って、当業者は、本明細書の記載があれば、過度な実験を行うことなく、使用すべき量等の実施形態を決定することができる。 The formulation procedure as a medicine such as a therapeutic drug and a preventive drug of the present invention is known in the art, and is described in, for example, the Japanese Pharmacopoeia, the US Pharmacopoeia, and the pharmacopoeia of other countries. Accordingly, those skilled in the art can determine the embodiment, such as the amount to be used, without undue experimentation as described herein.
　（一般技術）
　本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手法は、当該分野において周知であり慣用されるものであり、例えば、Ｓａｍｂｒｏｏｋ　Ｊ．ｅｔ　ａｌ．（１９８９）．Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒおよびその３ｒｄ　Ｅｄ．（２００１）；　Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９８７）．Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ　ａｎｄ　Ｗｉｌｅｙ−Ｉｎｔｅｒｓｃｉｅｎｃｅ；　Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９８９）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：　Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ　ａｎｄ　Ｗｉｌｅｙ−Ｉｎｔｅｒｓｃｉｅｎｃｅ；　Ｉｎｎｉｓ，Ｍ．Ａ．（１９９０）．ＰＣＲ　Ｐｒｏｔｏｃｏｌｓ：　Ａ　Ｇｕｉｄｅ　ｔｏ　Ｍｅｔｈｏｄｓ　ａｎｄ　Ａｐｐｌｉｃａｔｉｏｎｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ；　Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９９２）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：　Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ；　Ａｕｓｕｂｅｌ，Ｆ．Ｍ．　（１９９５）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：　Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｇｒｅｅｎｅ　Ｐｕｂ．Ａｓｓｏｃｉａｔｅｓ；　Ｉｎｎｉｓ，Ｍ．Ａ．ｅｔ　ａｌ．（１９９５）．ＰＣＲ　Ｓｔｒａｔｅｇｉｅｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ；Ａｕｓｕｂｅｌ，Ｆ．Ｍ．（１９９９）．Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ：　Ａ　Ｃｏｍｐｅｎｄｉｕｍ　ｏｆ　Ｍｅｔｈｏｄｓ　ｆｒｏｍ　Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｗｉｌｅｙ，ａｎｄ　ａｎｎｕａｌ　ｕｐｄａｔｅｓ；Ｓｎｉｎｓｋｙ，Ｊ．Ｊ．ｅｔ　ａｌ．（１９９９）．ＰＣＲ　Ａｐｐｌｉｃａｔｉｏｎｓ：　Ｐｒｏｔｏｃｏｌｓ　ｆｏｒ　Ｆｕｎｃｔｉｏｎａｌ　Ｇｅｎｏｍｉｃｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ、別冊実験医学「遺伝子導入＆発現解析実験法」羊土社、１９９７などに記載されており、これらは本明細書において関連する部分（全部であり得る）が参考として援用される。 (General technology)
Molecular biological techniques, biochemical techniques, and microbiological techniques used in this specification are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M.M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F .; M.M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M.M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M.M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, “Experimental Methods for Gene Transfer & Expression Analysis”, Yodosha, 1997, etc., which are related in this specification (may be all) Is incorporated by reference.
　人工的に合成した遺伝子を作製するためのＤＮＡ合成技術および核酸化学については、例えば、Ｇａｉｔ，Ｍ．Ｊ．（１９８５）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｓｙｎｔｈｅｓｉｓ：　Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，ＩＲＬ　Ｐｒｅｓｓ；　Ｇａｉｔ，Ｍ．Ｊ．（１９９０）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ　Ｓｙｎｔｈｅｓｉｓ：　Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，ＩＲＬ　Ｐｒｅｓｓ；　Ｅｃｋｓｔｅｉｎ，Ｆ．（１９９１）．Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ　ａｎｄ　Ａｎａｌｏｇｕｅｓ：Ａ　Ｐｒａｃｔｉｃａｌ　Ａｐｐｒｏａｃｈ，ＩＲＬ　Ｐｒｅｓｓ；Ａｄａｍｓ，Ｒ．Ｌ．ｅｔ　ａｌ．（１９９２）．Ｔｈｅ　Ｂｉｏｃｈｅｍｉｓｔｒｙ　ｏｆ　ｔｈｅ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，Ｃｈａｐｍａｎ＆　Ｈａｌｌ；　Ｓｈａｂａｒｏｖａ，Ｚ．ｅｔ　ａｌ．（１９９４）．Ａｄｖａｎｃｅｄ　Ｏｒｇａｎｉｃ　Ｃｈｅｍｉｓｔｒｙ　ｏｆ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，Ｗｅｉｎｈｅｉｍ；　Ｂｌａｃｋｂｕｒｎ，Ｇ．Ｍ．ｅｔａｌ．（１９９６）．Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　ｉｎ　Ｃｈｅｍｉｓｔｒｙ　ａｎｄ　Ｂｉｏｌｏｇｙ，Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ；Ｈｅｒｍａｎｓｏｎ，Ｇ．Ｔ．（Ｉ９９６）．Ｂｉｏｃｏｎｊｕｇａｔｅ　Ｔｅｃｈｎｉｑｕｅｓ，Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓなどに記載されており、これらは本明細書において関連する部分が参考として援用される。 For DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, see, for example, Gait, M. et al. J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Blackberry, G. Advanced Organic Chemistry of Nucleic Acids, Weinheim; M.M. etal. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T.A. (I996). Bioconjugate Technologies, Academic Press, etc., which are incorporated herein by reference for relevant portions.
　例えば、本明細書において、当該分野に知られる標準法によって、例えば自動化ＤＮＡ合成装置（Ｂｉｏｓｅａｒｃｈ、Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ等から市販されるものなど）の使用によって、本発明のオリゴヌクレオチドを合成することも可能である。例えば、Ｓｔｅｉｎら（Ｓｔｅｉｎ　ｅｔ　ａｌ．，１９８８，Ｎｕｃｌ．Ａｃｉｄｓ　Ｒｅｓ．１６：３２０９）の方法によって、ホスホロチオエート・オリゴヌクレオチドを合成することも可能であるし、調節孔ガラスポリマー支持体（Ｓａｒｉｎｅｔ　ａｌ．，１９８８，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８５：７４４８−７４５１）等の使用によって、メチルホスホネート・オリゴヌクレオチドを調製することも可能である。 For example, in the present specification, the oligonucleotide of the present invention can be synthesized by a standard method known in the art, for example, by using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.). is there. For example, it is possible to synthesize phosphorothioate oligonucleotides by the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res. 16: 3209), and control pore glass polymer supports (Sarinet al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451), etc., can also be used to prepare methylphosphonate oligonucleotides.
　本明細書において「または」は、文章中に列挙されている事項の「少なくとも１つ以上」を採用できるときに使用される。「もしくは」も同様である。本明細書において「２つの値の範囲内」と明記した場合、その範囲には２つの値自体も含む。 In this specification, “or” is used when “at least one or more” of the items listed in the sentence can be adopted. The same applies to “or”. In this specification, when “within the range of two values” is specified, the range includes the two values themselves.
　本明細書において引用された、科学文献、特許、特許出願などの参考文献は、その全体が、各々具体的に記載されたのと同じ程度に本明細書において参考として援用される。 References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as if they were specifically described.
　以上、本発明を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本発明を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本発明を限定する目的で提供したのではない。従って、本発明の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、特許請求の範囲によってのみ限定される。 As described above, the present invention has been described by showing preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.
　以下に実施例を記載する。必要な場合、以下の実施例で用いる動物の取り扱いは、全ての動物実験は、医薬基盤研究所及び大阪大学動物施設の機関ガイドラインに従って実施した。また、ヘルシンキ宣言に基づいて行った。試薬類は具体的には実施例中に記載した製品を使用したが、他メーカー（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ、和光純薬、ナカライ、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ、ＵＳＣＮ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ　ＩＮＣ等）の同等品でも代用可能である。 Examples are described below. When necessary, the animals used in the following examples were handled in accordance with institutional guidelines of the National Institute of Pharmaceutical Sciences and Osaka University animal facilities for all animal experiments. Also, based on the Declaration of Helsinki. Specifically, the reagents described in the examples were used as reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.
　（製造実施例）
　以下のＣｐＧ　ＯＤＮｓは、（株）ジーンデザインで合成された（下線はホスホロチオエート結合を示す）。 (Production Example)
The following CpG ODNs were synthesized by Gene Design Co., Ltd. (underlined indicates phosphorothioate linkage).
　特に、上記Ｋ３−ｄＡ４０（配列番号２）に加え、Ｋ３−ｄＡ３５（配列番号７）、Ｋ３−ｄＡ３０（配列番号６）、Ｋ３−ｄＡ２５（配列番号５）及び、Ｋ３−ｄＡ２０（配列番号４）の合成（表２）について記載する。 In particular, in addition to K3-dA40 (SEQ ID NO: 2), K3-dA35 (SEQ ID NO: 7), K3-dA30 (SEQ ID NO: 6), K3-dA25 (SEQ ID NO: 5), and K3-dA20 (SEQ ID NO: 4) Is described (Table 2).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（上記配列中のｓは、ヌクレオシド間のリン酸ジエステル結合がホスホロチオエート結合に置換されていることを示す。） (S in the above sequence indicates that the phosphodiester bond between nucleosides is substituted with a phosphorothioate bond.)
　本オリゴデオキシヌクレオチドは、常法である固相ホスホロアミダイト法（Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　ｉｎ　Ｃｈｅｍｉｓｔｒｙ　ａｎｄ　Ｂｉｏｌｏｇｙ，３．Ｃｈｅｍｉｃａｌ　ｓｙｎｔｈｅｓｉｓ（１９９０）ｅｄ．Ｇ．Ｍｉｃｈａｅｌ　Ｂｌａｃｋｂｕｒｎ　ａｎｄ　Ｍｉｃｈａｅｌ　Ｊ．Ｇａｉｔ．Ｏｘｆｏｒｄ　Ｕｎｉｖｅｒｓｉｔｙ　Ｐｒｅｓｓ）を用いて合成した。 This oligodeoxynucleotide can be obtained by using the conventional solid phase phosphoramidite method (Nucleic Acids in Chemistry and Biology, 3. Chemical synthesis (1990) ed. G. Michael Blackburn and Michael J. GaitOx. Synthesized.
　オブアルブミン（ＯＶＡ）は、生化学工業（株）から購入した。ＤＱ−ＯＶＡ、Ａｌｅｘａ４８８−ＯＶＡ、ＣＦＳＥ、及びＬｉｐｏｆｅｃｔａｍｉｎｅ２０００はＩｎｖｉｔｒｏｇｅｎから購入した。Ｈｏｅｃｈｓｔ３３２５８、ザイモザン及びカードランは、ＳＩＧＭＡから購入した。Ｚｙｍｏｓａｎ−ＤｅｐｌｅｔｅｄはＩｎｖｉｖｏｇｅｎから購入した。クロドロネートリポソームはＦｏｒｍｕＭａｘから購入した。インフルエンザスプリットプロダクトワクチン、ホルマリン不活化全ウイルス（ＷＩＶ）、及び精製インフルエンザウイルス（Ｈ１Ｎ１）は、以前に記載したように調製した（Ｋｏｙａｍａ，Ｓ．，ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ　ｔｒａｎｓｌａｔｉｏｎａｌ　ｍｅｄｉｃｉｎｅ　２，２５ｒａ２４（２０１０））。 Ovalbumin (OVA) was purchased from Seikagaku Corporation. DQ-OVA, Alexa488-OVA, CFSE, and Lipofectamine 2000 were purchased from Invitrogen. Hoechst 33258, zymozan and curdlan were purchased from SIGMA. Zymosan-Depleted was purchased from Invivogen. Clodronate liposomes were purchased from FormuMax. Influenza split product vaccine, formalin inactivated whole virus (WIV), and purified influenza virus (H1N1) were prepared as previously described (Koyama, S., et al., Science translational medicine 2, 25ra24 (2010) ).
　ＣｐＧ　ＯＤＮとＳＰＧとの複合体化（製造実施例図１）
　７．２２ｍｇのＫ３−ｄＡ４０を水（３．７ｍＬ）に溶解した。ＳＰＧ（三井製糖）１５ｍｇを０．２５Ｎ　ＮａＯＨ（１ｍＬ）に溶解した。１ｍＬの３３０ｍＭ　ＮａＨ２ＰＯ４をＤＮＡ溶液に加え、次にＳＰＧ溶液をＤＮＡ／ＮａＨ２ＰＯ４溶液に加え、４℃にて一晩維持することにより複合体化を完了した。モル比（ＭＳＰＧ／ＭＤＮＡ）は、０．２７に固定した。複合体の形成は、マイクロチップ電気泳動装置（ＳＨＩＭＡＤＺＵ：ＭｕｌｔｉＮＡ）によって確認した複合体の形成は、サイズ排除クロマトグラフィーを用い、ＣｐＧ　ＯＤＮの高分子量側へのシフトを、２６０ｎｍにおける吸収をモニタリングすることによって確認した（Ｓｙｓｔｅｍ：Ａｇｉｌｅｎｔ　１１００ｓｅｒｉｅｓ、Ｃｏｌｕｍｎ：Ａｓａｈｉｐａｋ　ＧＦ７Ｍ−ＨＱ（Ｓｈｏｄｅｘ）２本連結、Ｆｌｏｗ　ｒａｔｅ：０．８ｍＬ／ｍｉｎ、Ｂｕｆｆｅｒ：１０ｍＭ　ＥＤＴＡ　ＰＢＳ，ｐＨ７．４、Ｔｅｍｐｅｒａｔｕｒｅ：４０°Ｃ）。 Complexation of CpG ODN and SPG (Production Example Fig. 1)
7.22 mg of K3-dA40 was dissolved in water (3.7 mL). 15 mg of SPG (Mitsui Sugar) was dissolved in 0.25N NaOH (1 mL). Complexation was completed by adding 1 mL of 330 mM NaH2PO4 to the DNA solution and then adding the SPG solution to the DNA / NaH2PO4 solution and maintaining at 4 ° C. overnight. The molar ratio (MSPG / MDNA) was fixed at 0.27. The formation of the complex was confirmed by a microchip electrophoresis apparatus (SHIMADZU: MultiNA). The formation of the complex was performed by using size exclusion chromatography and monitoring the shift of CpG ODN toward high molecular weight at 260 nm. (System: Agilent 1100 series, Column: Asahipak GF7M-HQ (Shodex) 2 ligation, Flow rate: 0.8 mL / min, Buffer: 10 mM EDTA PBS, pH 7.4, Temperature: 40 ° C.).
　（実施例に使用するための準備）
　以下の実施例において、強い腫瘍縮小を誘発する腫瘍微小環境における貪食細胞を標的とするナノ粒子状ＴＬＲ９アゴニストによる全身性の単剤治療が可能であることを示した。 (Preparation for use in examples)
In the examples below, it was shown that systemic monotherapy with nanoparticulate TLR9 agonists targeting phagocytic cells in a tumor microenvironment that induces strong tumor shrinkage is possible.
　（材料および方法）
　以下に、本実施例で用いられる試薬、材料、動物、細胞およびその方法について説明する。適宜、各実施例においても補充して説明する。 (Materials and methods)
Hereinafter, reagents, materials, animals, cells and methods used in this example will be described. The description will be supplemented in each embodiment as appropriate.
　（動物および試薬）
　６週齢の雌性Ｃ５７ＢＬ／６ＪマウスをＮｉｈｏｎ　ＣＬＥＡから購入した。Ｉｌ１２ｐ４０欠損マウスおよびＢａｔｆ３欠損マウスをＪａｃｋｓｏｎ　Ｌａｂｏｒａｔｏｒｙから購入した。Ｉｆｎａｒ２欠損マウス、Ｍｙｄ８８欠損マウスおよびＤｅｃｔｉｎ−１欠損マウスは以前記載した通りである（Ｋｏｂｉｙａｍａ，Ｋ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１１１，３０８６−３０９１（２０１４））。全ての動物実験を、医薬基盤研究所の機関ガイドラインに従って行った。Ｋ３はＧｅｎｅ　Ｄｅｓｉｇｎにより合成された。オバルブミン（ＯＶＡ）を生化学工業から購入した。 (Animals and reagents)
Six week old female C57BL / 6J mice were purchased from Nihon CLEA. Il12p40-deficient mice and Batf3-deficient mice were purchased from Jackson Laboratory. Ifnar2-deficient mice, Myd88-deficient mice, and Dectin-1-deficient mice are as previously described (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014 )). All animal experiments were performed according to the Institute of Pharmaceutical Sciences Institute guidelines. K3 was synthesized by Gene Design. Ovalbumin (OVA) was purchased from Seikagaku.
　（細胞株）
　ＥＬ４およびＯＶＡ発現ＥＬ４（ＥＧ７）は、Ｃ５７ＢＬ／６Ｊマウスの胸腺腫細胞株であり、ＡＴＣＣから購入した。Ｂ１６（黒色腫）をＪａｐａｎｅｓｅ　Ｃｏｌｌｅｃｔｉｏｎ　ｏｆ　Ｒｅｓｅａｒｃｈ　Ｂｉｏｒｅｓｏｕｒｓｅｓから購入した。Ｂ１６Ｆ１０（黒色腫）を理研細胞バンクから購入し、ＭＣ３８（結腸がん）は、Ｆ．ＪＡＭＥＳ　Ｐｒｉｍｕｓ博士により提供された。Ｐａｎ０２（膵臓がん）をＪａｃｋｓｏｎ’ｓ　Ｌａｂｏｒａｔｏｒｙから購入した。ＥＬ４、ＥＧ７、ＭＣ３８、およびＰａｎ０２を、完全ＲＰＭＩ（１０％（ｖ／ｖ）ウシ胎児血清（ＦＢＳ）、ペニシリン、およびストレプトマイシンが追加されたＲＰＭＩ　１６４０）で培養した。Ｂ１６およびＢ１６Ｆ１０を、完全ＤＭＥＭ（１０％（ｖ／ｖ）ウシ胎児血清（ＦＢＳ）、ペニシリン、およびストレプトマイシンが追加されたＤＭＥＭ）で培養した。 (Cell line)
EL4 and OVA expressing EL4 (EG7) is a thymoma cell line of C57BL / 6J mice and was purchased from ATCC. B16 (melanoma) was purchased from the Japan Collection of Research Bioresources. B16F10 (melanoma) was purchased from Riken Cell Bank and MC38 (colon cancer) was Provided by Dr. JAMES Primus. Pan02 (pancreatic cancer) was purchased from Jackson's Laboratory. EL4, EG7, MC38, and Pan02 were cultured in complete RPMI (RPMI 1640 supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin, and streptomycin). B16 and B16F10 were cultured in complete DMEM (DMEM supplemented with 10% (v / v) fetal bovine serum (FBS), penicillin, and streptomycin).
　（腫瘍実験および治療方法）
　ＥＧ７、ＥＬ４、Ｂ１６、Ｂ１６Ｆ１０、およびＭＣ３８細胞（１０％マトリゲル／ＰＢＳ中に５×１０６細胞／ｍＬで１００μｌ）を、マウスの右側腹部の皮下（ｓ．ｃ）に接種した。腫瘍サイズを、腫瘍の長さ（Ｌ）、幅（Ｗ）、および高さ（Ｈ）で測定し、腫瘍体積（Ｖ）をＶ＝Ｌ×Ｗ×Ｈとして計算した。腫瘍内注射（ｉ．ｔ．）では、腫瘍部位に直接注射した。ＣｐＧ治療は、腫瘍体積が約１００ｍｍ３に達してから開始し、その時期は、ＥＧ７およびＢ１６Ｆ１０の接種の７日後、Ｂ１６の接種の１０日後、ならびにＭＣ３８の接種の１４日後であった。腫瘍保有マウスを、Ｋ３（３０μｇ）またはＫ３−ＳＰＧ（１０μｇ）で１日おきに３回処置した。 (Tumor experiment and treatment method)
EG7, EL4, B16, B16F10, and MC38 cells (100 μl at 5 × 10 6 cells / mL in 10% Matrigel / PBS) were inoculated subcutaneously (sc) in the right flank of mice. Tumor size was measured by tumor length (L), width (W), and height (H), and tumor volume (V) was calculated as V = L × W × H. Intratumoral injection (it) was injected directly into the tumor site. CpG treatment began when the tumor volume reached approximately 100 mm 3 , which was 7 days after EG7 and B16F10 inoculation, 10 days after B16 inoculation, and 14 days after MC38 inoculation. Tumor-bearing mice were treated 3 times every other day with K3 (30 μg) or K3-SPG (10 μg).
　（Ｐａｎ０２の腹膜播種モデル）
　Ｐａｎ０２の腹膜播種モデルにおいて、１×１０６個のＰａｎ０２細胞（ＰＢＳ中に１×１０７細胞／ｍＬで１００μｌ）を、腹腔内に注射した。ＣｐＧ治療を接種１１日後に開始し、全ての腫瘍小結節を２１日目にマウスの腹膜から摘出し、その後これらの重量（ｇ）を測定した。ＣｐＧ治療での投与量は上記のとおりである。 (Pan02 peritoneal seeding model)
In the Pan02 peritoneal seeding model, 1 × 10 6 Pan02 cells (100 μl at 1 × 10 7 cells / mL in PBS) were injected intraperitoneally. CpG treatment was started 11 days after inoculation and all tumor nodules were excised from the peritoneum of mice on day 21 and their weight (g) was then measured. The dosage for CpG treatment is as described above.
　（ｉｎ　ｖｉｖｏイメージング実験）
　Ｋ３およびＫ３−ＳＰＧの局在を評価するために、Ｃ５７ＢＬ／６マウスに、ＥＧ７を０日目にｓ．ｃ．接種し、ＰＢＳ（コントロール）、Ａｌｅｘａ　６４７−Ｋ３（３０μｇ）、またはＡｌｅｘａ　６４７−Ｋ３−ＳＰＧ（１０μｇ）を１２日目にｉ．ｖ．投与した。投与１時間後、マウスをＩＶＩＳ（登録商標）Ｌｕｍｉｎａ　Ｉｍａｇｉｎｇ　Ｓｙｓｔｅｍおよび分析ソフトウェア（Ｖｅｒ．２．６、Ｘｅｎｏｇｅｎ）で分析し、相対的蛍光で測定されたイメージを、表面放射輝度の物理単位（光子／秒／ｃｍ２／ｓｒ）に変換した。ｉｎ　ｖｉｖｏで標識されたＣＤ８＋Ｔ細胞を検出するために、脾細胞を、７、９、１１日目にＫ３−ＳＰＧで処置されたか、または処置されていない、ＥＧ７を保有するＣ５７ＢＬ／６マウスまたはＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウスから１４日目に回収した。脾細胞の懸濁後、赤血球をＡＣＫ溶解緩衝液（１５０ｍＭ　ＮＨ４Ｃｌ、１０ｍＭ　ＫＨＣＯ３、０．１ｍＭ　Ｎａ２ＥＤＴＡ）で溶解し、細胞を完全ＲＰＭＩ中に維持した。ＣＤ８α＋Ｔ細胞をＭＡＣＳ（Ｍｉｌｔｅｎｙｉ　Ｂｉｏｔｅｃ）によりソートした。ＣＤ８α＋Ｔ細胞を陰性選択の方法でソートした。その後、ソートされたＣＤ８α＋Ｔ細胞を、Ｘｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で染色した。染色されたＣＤ８α＋Ｔ細胞を、１４日目にレシピエントマウス（０日目にＥＧ７を接種し、７、９、１１日目にＫ３−ＳＰＧでｉ．ｖ．処置されたか、または処置されていないＣ５７ＢＬ／６マウスまたはＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウス）に移した。染色した細胞を移して２４時間後に、マウスをＩＶＩＳ（登録商標）Ｌｕｍｉｎａ　Ｉｍａｇｉｎｇ　Ｓｙｓｔｅｍ（Ｖｅｒ．２．６）で分析した。目的の領域を腫瘍領域に集約し、蛍光強度をＬｉｖｉｎｇ　Ｉｍａｇｅ　Ｓｏｆｔｗａｒｅ（Ｖｅｒ．２．６、Ｘｅｎｏｇｅｎ）で分析した。 (In vivo imaging experiment)
To assess the localization of K3 and K3-SPG, C57BL / 6 mice were treated with EG7 at day 0. c. Inoculate and receive PBS (control), Alexa 647-K3 (30 μg), or Alexa 647-K3-SPG (10 μg) i. v. Administered. One hour after dosing, mice were analyzed with IVIS® Lumina Imaging System and analysis software (Ver. 2.6, Xenogen), and images measured with relative fluorescence were measured in physical units (photons / photon / surface radiance). Second / cm 2 / sr). To detect CD8 + T cells labeled in vivo, splenocytes were treated with K3-SPG on days 7, 9, 11 or untreated, C57BL / 6 mice carrying EG7 Or harvested on day 14 from Il12p40-Ifnar2 double knockout mice. After spleen cell suspension, erythrocytes were lysed with ACK lysis buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA) and cells were maintained in complete RPMI. CD8α + T cells were sorted by MACS (Miltenyi Biotec). CD8α + T cells were sorted by the negative selection method. The sorted CD8α + T cells were then stained with Xenolight DiR®. Stained CD8α + T cells were injected or treated with recipient mice (EG7 on day 0, iv7 on days 0, and K3-SPG on days 7, 9, 11). C57BL / 6 mice or Il12p40-Ifnar2 double knockout mice). Twenty-four hours after transferring the stained cells, the mice were analyzed with the IVIS® Lumina Imaging System (Ver. 2.6). The region of interest was aggregated into the tumor region and the fluorescence intensity was analyzed with Living Image Software (Ver. 2.6, Xenogen).
　（免疫組織化学）
　Ｃ５７ＢＬ／６Ｊマウス（６−８週齢、雌、日本クレア）に、Ａｌｅｘａ　６４７−Ｋ３（３０μｇ）、Ａｌｅｘａ　６４７−Ｋ３−ＳＰＧ（１０μｇ）、およびデキストラン−ＰＥ（２０μｇ）を尾静脈からｉ．ｖ．注射した。腫瘍を注射後１時間後に回収し、凍結切片を、４％（ｗ／ｖ）パラホルムアルデヒドで１０分間固定し、Ｈｏｅｃｈｓｔ　３３２５８と一緒に抗ＣＤ３ｅ抗体、抗ＣＤ８β抗体で染色した。細胞をＯｌｙｍｐｕｓ　ＩＸ８１システムを使用して撮影した。イメージデータをＭｅｔａＭｏｒｐｈで分析した。 (Immunohistochemistry)
C57BL / 6J mice (6-8 weeks old, female, CLEA Japan) were treated with Alexa 647-K3 (30 μg), Alexa 647-K3-SPG (10 μg), and dextran-PE (20 μg) i.e. from the tail vein. v. Injected. Tumors were collected 1 hour after injection and frozen sections were fixed with 4% (w / v) paraformaldehyde for 10 minutes and stained with anti-CD3e and anti-CD8β antibodies together with Hoechst 33258. Cells were photographed using an Olympus IX81 system. Image data was analyzed with MetaMorph.
　（枯渇実験）
　食細胞（樹状細胞およびマクロファージ）を枯渇させるために、Ｃ５７ＢＬ／６マウスに、クロドロネートリポソームまたはコントロールリポソーム（１００ｎｍ）（片山化学）をＥＧ７の接種の５日後にｉ．ｖ．注射した。ＣＤ８＋Ｔ細胞を枯渇させるために、２００μｇの抗ＣＤ８α抗体を、ＥＧ７接種の６日後および１３日後に尾静脈にｉ．ｖ．注射した。 (Depletion experiment)
In order to deplete phagocytic cells (dendritic cells and macrophages), C57BL / 6 mice were treated with clodronate liposomes or control liposomes (100 nm) (Katayama Chemical) i. v. Injected. To deplete CD8 + T cells, 200 μg of anti-CD8α antibody was administered i.v. to the tail vein 6 and 13 days after EG7 inoculation. v. Injected.
　（脾細胞の分析）
　脾細胞を、７、９、１１日目にＫ３−ＳＰＧでｉ．ｖ．処置されたか、処置されていない、ＥＧ７を保有するＣ５７ＢＬ／６マウスまたはＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウスから１４日目に回収した。脾細胞の調製後、赤血球をＡＣＫ溶解緩衝液で溶解し、細胞を完全ＲＰＭＩで維持した。脾細胞をＨ−２Ｋｂ　ＯＶＡテトラマー（ＭＢＬ）、抗ＣＤ８α抗体（ＫＴ１５）、抗ＴＣＲβ抗体（Ｈ５７−５９７）、抗ＣＤ６２Ｌ抗体（ＭＥＬ−１４）、および抗ＣＤ４４抗体（ＩＭ７）、ならびに７−アミノアクチノマイシンＤ（７ＡＡＤ）で染色した。ＯＶＡテトラマー＋ＣＤ４４＋ＣＤ８α＋ＴＣＲβ＋の細胞数を、フローサイトメトリーで決定した。他の実験については、調製された脾細胞を、抗ＣＤ４５抗体、抗ＣＤ３ｅ抗体、抗ＣＤ８α抗体、および抗ＣＤ１１ａ抗体と共にインキュベートし、その後フローサイトメトリーで分析した。 (Analysis of splenocytes)
Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Recovered on day 14 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained with complete RPMI. Splenocytes were isolated from H-2K b OVA tetramer (MBL), anti-CD8α antibody (KT15), anti-TCRβ antibody (H57-597), anti-CD62L antibody (MEL-14), and anti-CD44 antibody (IM7), and 7-amino. Stained with actinomycin D (7AAD). The cell number of OVA tetramer + CD44 + CD8α + TCRβ + was determined by flow cytometry. For other experiments, prepared splenocytes were incubated with anti-CD45 antibody, anti-CD3e antibody, anti-CD8α antibody, and anti-CD11a antibody and then analyzed by flow cytometry.
　（ＣＤ４５陰性細胞のアッセイおよび免疫化）
　脾細胞を、７、９、１１日目にＫ３−ＳＰＧでｉ．ｖ．処置されたか、処置されていない、ＥＧ７を保有するＣ５７ＢＬ／６マウスまたはＩｌ１２ｐ４０−Ｉｆｎａｒ２ダブルノックアウトマウスから１２日目に回収した。脾細胞の調製後、赤血球をＡＣＫ溶解緩衝液で溶解し、細胞を完全ＲＰＭＩ中に維持した。脾細胞を抗ＣＤ４５抗体（ＡＰＣ）で染色し、ＣＤ４５−細胞の数をフローサイトメトリーで決定した。さらに、アポトーシス細胞、ネクローシス細胞、およびＣＤ４５陰性生細胞の集団を、ＰＩおよびＨｏｅｃｈｓｔ　３３３４２で染色し、その後フローサイトメトリーで分析した。次に、ＣＤ４５−細胞を、Ｋ３−ＳＰＧで処置された腫瘍保有Ｃ５７ＢＬ／６マウスから、ＩＮＦＬＵＸ（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅ）によってソートした。 (Assay and immunization of CD45 negative cells)
Splenocytes were injected i.p. with K3-SPG on days 7, 9, 11. v. Harvested at day 12 from treated or untreated C57BL / 6 mice carrying EG7 or Il12p40-Ifnar2 double knockout mice. After preparation of splenocytes, erythrocytes were lysed with ACK lysis buffer and the cells were maintained in complete RPMI. Splenocytes were stained with anti-CD45 antibody (APC) and the number of CD45 − cells was determined by flow cytometry. In addition, populations of apoptotic cells, necrotic cells, and live CD45 negative cells were stained with PI and Hoechst 33342 and then analyzed by flow cytometry. Next, CD45 − cells were sorted by INFLUX (BD Bioscience) from tumor bearing C57BL / 6 mice treated with K3-SPG.
　（ワクチン接種モデル）
　Ｃ５７ＢＬ／６マウスに、５×１０５個のＣＤ４５−細胞を−７日目にｉ．ｖ．投与した。免疫化７日後、マウスに５×１０５個のＥＧ７細胞を０日目にｓ．ｃ．接種した。 (Vaccination model)
C57BL / 6 mice were challenged with 5 × 10 5 CD45 − cells on day −7. v. Administered. Seven days after immunization, mice were challenged with 5 × 10 5 EG7 cells on day 0. c. Vaccinated.
　（サイトカインの測定）
　マウスＩＬ−１２ｐ４０、マウスＩＬ−１３、およびヒトＩＦＮγのレベルを、Ｒ＆ＤのＥＬＩＳＡキットを用いて測定した。 (Measurement of cytokines)
Mouse IL-12p40, mouse IL-13, and human IFNγ levels were measured using an R & D ELISA kit.
　（統計分析）
　Ｍａｎｎ−ＷｈｉｔｎｅｙのＵ検定、Ｓｔｕｄｅｎｔのｔ検定またはＢｏｎｆｅｒｒｏｎｉの多重比較検定を含む一元分散分析を、統計分析に用いた（＊ｐ＜０．０５；＊＊ｐ＜０．０１；＊＊＊ｐ＜０．００１）。統計分析をＧｒａｐｈＰａｄ　Ｐｒｉｓｍ　ｓｏｆｔｗａｒｅ（Ｌａ　Ｊｏｌｌａ、ＣＡ、ＵＳＡ）を用いて行った。 (Statistical analysis)
One-way analysis of variance including Mann-Whitney U test, Student's t test or Bonferroni's multiple comparison test was used for statistical analysis ( * p <0.05; ** p <0.01; *** p < 0.001). Statistical analysis was performed using GraphPad Prism software (La Jolla, CA, USA).
　（実施例１：Ｋ３−ＳＰＧの静脈内注射は、腫瘍抗原を全く追加しなくても強い腫瘍成長の抑制を誘発する）
　本実施例では、Ｋ３−ＳＰＧの静脈内注射により、腫瘍抗原を全く追加しなくても強い腫瘍成長の抑制が誘発されることを実証した。 (Example 1: Intravenous injection of K3-SPG induces strong tumor growth inhibition without any additional tumor antigen)
In this example, it was demonstrated that intravenous injection of K3-SPG induces strong tumor growth inhibition without any additional tumor antigen.
　（ＥＧ７（ＯＶＡ発現マウス胸腺腫細胞株）モデルでの実験）
　Ｃ５７ＢＬ／６マウスに、ＥＧ７（ＯＶＡ発現マウス胸腺腫細胞株）を右側腹部に０日目に接種し、尾基部付近の皮下（ｉ．ｄ．）投与、腫瘍内（ｉ．ｔ．）投与、または静脈内（ｉ．ｖ．）投与の３つの異なる経路を介して、ＰＢＳ、および等モル量のＫ３（３０μｇ）またはＫ３−ＳＰＧ（１０μｇ）で３回処置した（接種の７、９、１１日後）。腫瘍サイズを２３日目まで２~３日毎に測定した。 (Experiment with EG7 (OVA expressing mouse thymoma cell line) model)
C57BL / 6 mice were inoculated with EG7 (OVA expressing mouse thymoma cell line) on the right flank on day 0, administered subcutaneously (id) near the tail base, intratumoral (it), Or three times with PBS and equimolar amounts of K3 (30 μg) or K3-SPG (10 μg) via three different routes of intravenous (iv) administration (7, 9, 11 of inoculation) days after). Tumor size was measured every 2-3 days until day 23.
　（結果）
　結果は図２（Ａ~Ｂ）以下に表す。ＰＢＳの群（コントロール）において、腫瘍成長はいずれの投与経路を介しても抑制されなかった（図２ａ、ｂ、ｃ（図２Ａ））。Ｋ３処置において、腫瘍縮小がｉ．ｔ．でのみ観察されたが、他の経路においては観察されなかった（図２ｄ、ｅ、ｆ（図２Ａ））。Ｋ３−ＳＰＧ処置において、ｉ．ｔ．およびｉ．ｖ．の両方で強い腫瘍縮小が観察されたが、ｉ．ｄ．投与は、腫瘍成長に対する影響を示さなかった（図２ｇ、ｈ、ｉ（図２Ａ））。コントロール、Ｋ３およびＫ３−ＳＰＧを比較して示した図を図（図２ａ、ｄ、ｇ（図２Ａ））に示す。 (result)
The results are shown in FIG. 2 (AB) and below. In the PBS group (control), tumor growth was not inhibited via any route of administration (FIGS. 2a, b, c (FIG. 2A)). In K3 treatment, tumor shrinkage is i. t. Was observed only in, but not in other routes (FIGS. 2d, e, f (FIG. 2A)). In K3-SPG treatment, i. t. And i. v. Strong tumor shrinkage was observed in both cases, i. d. Administration showed no effect on tumor growth (Figure 2g, h, i (Figure 2A)). The figure which compared and showed control, K3, and K3-SPG is shown in a figure (FIG. 2 a, d, g (FIG. 2A)).
　従来技術では、がんに対する全身性ＣｐＧ　ＯＤＮ治療での多くの試みは成功していなかった（Ｌｏｕ，Ｙ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｔｈｅｒａｐｙ（Ｈａｇｅｒｓｔｏｗｎ，Ｍｄ．：　１９９７）３４，２７９−２８８　（２０１１）；Ｎｉｅｒｋｅｎｓ，Ｓ．，ｅｔ　ａｌ．ＰＬｏＳ　Ｏｎｅ　４，ｅ８３６８　（２００９））ことから、Ｋ３−ＳＰＧでのｉ．ｖ．単剤治療は、腫瘍成長を強く抑制し得るという事実は予測できなかったことであり、この点で本発明は予想外の効果を生じていることが実証された。 In the prior art, many attempts at systemic CpG ODN treatment for cancer have not been successful (Lou, Y., et al. Journal of immunotherapy (Hagetown, Md .: 1997) 34, 279-288 (2011). Nierkens, S., et al. PLoS One 4, e8368 (2009)), i. v. The fact that single agent treatment can strongly suppress tumor growth was unpredictable, and in this respect it was demonstrated that the present invention had an unexpected effect.
　（他の腫瘍細胞株での実験）
　このＫ３−ＳＰＧ全身性単剤治療の潜在性を調査するために、他の腫瘍細胞株もまた、ＥＧ７モデルにおいて使用された同様のプロトコルで試験した。 (Experiment with other tumor cell lines)
To investigate the potential of this K3-SPG systemic monotherapy, other tumor cell lines were also tested with similar protocols used in the EG7 model.
　（結果）
　Ｋ３−ＳＰＧの静脈内投与は、黒色腫（Ｂ１６およびＢ１６Ｆ１０）および結腸がん（ＭＣ３８）の成長も抑制した（図２ｊ、ｋ、ｌ（図２Ｂ））。本発明者らは、さらにより臨床的悪性度の高い腫瘍播種性モデルを作製することによって試験した。マウス膵臓腫瘍株、Ｐａｎ０２（１×１０６細胞）を、腹腔内（「ｉ．ｐ．」とも称する）に接種し、その後、Ｋ３またはＫ３−ＳＰＧによる治療（１日おきに３回）を接種の１１日後に開始した。２１日目に全てのマウスを屠殺し、腹腔の腫瘍の総重量を評価した（図２ｍ（図２Ｂ））。腫瘍成長は、Ｋ３−ＳＰＧ　ｉ．ｖ．処置群において顕著に抑制されたが、Ｋ３　ｉ．ｐ．およびＫ３−ＳＰＧ　ｉ．ｐ．処置群においては抑制されなかった（図２ｍ（図２Ｂ））。それに応じて、顕著な生存の延長が、Ｋ３−ＳＰＧ　ｉ．ｖ．処置群において観察されたが、Ｋ３　ｉ．ｖ．群では観察されなかった（図２ｎ（図２Ｂ））。これらの結果は、Ｋ３−ＳＰＧの全身性ｉ．ｖ．投与が、多くの異なるがんに対する有望な単剤治療であり、いずれの腫瘍ペプチドおよび抗原もさらに必要としないことを実証するものである。 (result)
Intravenous administration of K3-SPG also suppressed the growth of melanoma (B16 and B16F10) and colon cancer (MC38) (Figure 2j, k, l (Figure 2B)). We tested by creating a tumor dissemination model with even higher clinical malignancy. Mouse pancreatic tumor line, Pan02 (1 × 10 6 cells) is inoculated intraperitoneally (also referred to as “ip”) followed by K3 or K3-SPG treatment (3 times every other day) 11 days later. On day 21, all mice were sacrificed and the total weight of the abdominal tumor was assessed (FIG. 2m (FIG. 2B)). Tumor growth is determined by K3-SPG i. v. Although significantly suppressed in the treatment group, K3 i. p. And K3-SPG i. p. It was not suppressed in the treatment group (FIG. 2m (FIG. 2B)). In response, a significant prolongation of survival was observed in K3-SPG i. v. Although observed in the treatment group, K3 i. v. Not observed in the group (FIG. 2n (FIG. 2B)). These results indicate that the systemic i. v. It demonstrates that administration is a promising monotherapy for many different cancers and does not require any further tumor peptides and antigens.
　（実施例２：Ｋ３−ＳＰＧは腫瘍微小環境において食細胞を標的とした）
　次に、本発明者らは、Ｋ３−ＳＰＧの腫瘍微小環境におけるメカニズムを解明した。 (Example 2: K3-SPG targeted phagocytic cells in tumor microenvironment)
Next, the present inventors elucidated the mechanism of K3-SPG in the tumor microenvironment.
　Ｋ３−ＳＰＧは、約３０ｎｍのサイズのナノ粒子を形成する（Ｋｏｂｉｙａｍａ，Ｋ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１１１，３０８６−３０９１　（２０１４））。本発明者らは、Ｋ３−ＳＰＧは、腫瘍までの薬物送達システムを介して機能すると仮定した（Ｎａ，Ｊ．Ｈ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｃｏｎｔｒｏｌｌｅｄ　ｒｅｌｅａｓｅ　：　ｏｆｆｉｃｉａｌ　ｊｏｕｒｎａｌ　ｏｆ　ｔｈｅ　Ｃｏｎｔｒｏｌｌｅｄ　Ｒｅｌｅａｓｅ　Ｓｏｃｉｅｔｙ　１６３，２−９　（２０１２）；Ｐｅｔｒｏｓ，Ｒ．Ａ．ｅｔ　ａｌ．Ｎａｔ　Ｒｅｖ　Ｄｒｕｇ　Ｄｉｓｃｏｖ　９，６１５−６２７　（２０１０）；Ｐａｎｔｅ，Ｎ．ｅｔ　ａｌ．Ｍｏｌｅｃｕｌａｒ　ｂｉｏｌｏｇｙ　ｏｆ　ｔｈｅ　ｃｅｌｌ　１３，４２５−４３４　（２００２）；Ｄａｖｉｓ，Ｍ．Ｅ．，ｅｔ　ａｌ．Ｎａｔ　Ｒｅｖ　Ｄｒｕｇ　Ｄｉｓｃｏｖ　７，７７１−７８２　（２００８）；Ｆａｒｏｋｈｚａｄ，Ｏ．Ｃ．ｅｔ　ａｌ．ＡＣＳ　Ｎａｎｏ　３，１６−２０（２００９））。 K3-SPG forms nanoparticles with a size of about 30 nm (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)). The present inventors hypothesized that K3-SPG functions via a drug delivery system up to the tumor (Na, JH, et al. Journal of controlled release: official journal of the Controlled Release Society 3, -9 (2012); Petros, RA et al. Nat Rev Drug Discov 9, 615-627 (2010); Pante, N. et al. Molecular biology of the cell 13,425-434 (2002); , ME, et al. Nat Rev Drug Discov 7, 771-782 (2008); O.C.et al.ACS Nano 3,16-20 (2009)).
　（蛍光標識イメージング）
　ｉｎ　ｖｉｖｏでの分布を試験するために、Ｋ３およびＫ３−ＳＰＧを蛍光標識した。ＥＧ７腫瘍保有マウスにＰＢＳ、Ａｌｅｘａ６４７−Ｋ３（３０μｇ）、またはＡｌｅｘａ６４７−Ｋ３−ＳＰＧ（１０μｇ）をｉ．ｖ．注射し、その後、蛍光の分布をｉｎ　ｖｉｖｏイメージングシステム（ＩＶＩＳ）によって試験した。 (Fluorescent labeling imaging)
To test the distribution in vivo, K3 and K3-SPG were fluorescently labeled. EG7 tumor-bearing mice were given PBS, Alexa647-K3 (30 μg), or Alexa647-K3-SPG (10 μg) i. v. After injection, the distribution of fluorescence was examined by an in vivo imaging system (IVIS).
　結果を図４以下に示す。 The results are shown in FIG.
　ＩＶＩＳイメージングは、Ｋ３ではなくＫ３−ＳＰＧが、ｉ．ｖ．投与１時間後で腫瘍部位に蓄積したことを明らかにした（図４ａ）。腫瘍におけるＫ３−ＳＰＧの蓄積は、ＣｐＧ単剤治療の腫瘍縮小の有効性によく関連していることが観察された（図２）。免疫組織化学（ＩＨＣ）試験において、本発明者らは、腫瘍微小環境においてＡｌｅｘａ６４７−Ｋ３を検出することができなかった（図４ｂ）。他方で、Ａｌｅｘａ６４７−Ｋ３−ＳＰＧは、腫瘍領域で観察された（図４ｃ）。本発明者らは、２４時間後にはＩＨＣでのＡｌｅｘａ６４７シグナルを検出することができなかった。ＥＧ７細胞は、その表面上にＣＤ３ｅを発現するが（なぜなら、ＥＧ７は胸腺腫細胞株に由来するため）、Ｋ３−ＳＰＧはＣＤ３ｅに会合せず、これはＫ３−ＳＰＧは非腫瘍細胞によって取り込まれたことを示した。ナノ粒子は、マクロファージおよび樹状細胞（ＤＣ）などの食細胞によって取り込まれることを選択し、これらの細胞は、ｉｎ　ｖｉｖｏにおいてＴＲＩＴＣ−デキストランによって標識され得る。そのため、本発明者は、蛍光染色されたＫ３、Ｋ３−ＳＰＧ、またはＳＰＧと一緒にＴＲＩＴＣ−デキストランを静脈内注射し、ＩＨＣによるそれらの共存について試験した（図４ｄ、ｅ、ｆ）。ｉ．ｖ．注射の１時間後、デキストランは、全試料の腫瘍領域の中で観察され（図４ｄ、ｅ、ｆ）、これは腫瘍微小環境が食細胞を含んでいることを示している。以前の結果と一貫して、Ａｌｅｘａ６４７−Ｋ３は腫瘍内で観察されなかった（図４ｄ）。腫瘍内で観察された約５０％のＡｌｅｘａ６４７−Ｋ３−ＳＰＧおよびＦＩＴＣ−ＳＰＧは、ＴＲＩＴＣ−デキストラン陽性細胞と共存し（図４ｅ、ｆ、ｇ）、これはＫ３−ＳＰＧが、腫瘍微小環境において食細胞によって取り込まれることを示している。一部のＫ３−ＳＰＧはデキストランと会合せず、本発明者らは、血管透過性・滞留性亢進（ＥＰＲ）効果を介して、それらは腫瘍組織内の空間に受動的に蓄積したと推測している。Ｋ３−ＳＰＧ　ｉ．ｖ．処置に対する食細胞の重要性を試験するために、本発明者らは、クロドロネートリポソームを静脈内注射した。本発明者らは、通常２００~３００ｎｍのリポソームではなくて１００ｎｍのクロドロネートリポソームを使用して、これを注射することにより腫瘍内の食細胞を枯渇させ（Ｐａｎｔｅ，Ｎ．ｅｔ　ａｌ．Ｍｏｌｅｃｕｌａｒ　ｂｉｏｌｏｇｙ　ｏｆ　ｔｈｅ　ｃｅｌｌ　１３，４２５−４３４　（２００２）；Ｐａｎｔｅ，Ｎ．ｅｔ　ａｌ．Ｍｏｌｅｃｕｌａｒ　ｂｉｏｌｏｇｙ　ｏｆ　ｔｈｅ　ｃｅｌｌ　１３，４２５−４３４　（２００２））、この注射により、腫瘍におけるＦ４／８０陽性細胞が２日でほとんど枯渇した（図５）。腫瘍保有マウスに、クロドロネートリポソームを５日目に（最初のＫ３−ＳＰＧ処置の２日前）注射するか、または注射せず、図２（Ａ~Ｂ）と同様にマウスをＫ３−ＳＰＧで処置した。先にクロドロネートリポソームを注射した場合、Ｋ３−ＳＰＧ媒介腫瘍成長の抑制を顕著に相殺し（ｐ＜０．０５）（図４ｈ）、他方で、クロドロネートリポソームの注射それ自体は、ＰＢＳ処置マウスと比較して腫瘍成長に影響を与えなかった。これらの結果は、Ｋ３−ＳＰＧが、腫瘍微小環境における食細胞を標的とし、Ｋ３−ＳＰＧの抗腫瘍効果は、Ｋ３−ＳＰＧの腫瘍微小環境における食細胞への取り込みにほとんど依存していることを示している。 IVIS imaging uses K3-SPG instead of K3, i. v. It was revealed that it accumulated at the tumor site 1 hour after administration (FIG. 4a). It was observed that the accumulation of K3-SPG in the tumor is well related to the effectiveness of CpG monotherapy treatment for tumor shrinkage (FIG. 2). In immunohistochemistry (IHC) studies, we were unable to detect Alexa647-K3 in the tumor microenvironment (FIG. 4b). On the other hand, Alexa647-K3-SPG was observed in the tumor area (FIG. 4c). We were unable to detect Alexa647 signal on IHC after 24 hours. EG7 cells express CD3e on their surface (because EG7 is derived from a thymoma cell line), but K3-SPG does not associate with CD3e, which is taken up by non-tumor cells. It showed that. Nanoparticles choose to be taken up by phagocytic cells such as macrophages and dendritic cells (DC), and these cells can be labeled with TRITC-dextran in vivo. Therefore, the inventor injected TRITC-dextran intravenously with fluorescently stained K3, K3-SPG, or SPG and tested for their coexistence by IHC (FIGS. 4d, e, f). i. v. One hour after injection, dextran was observed in the tumor area of all samples (FIGS. 4d, e, f), indicating that the tumor microenvironment contains phagocytes. Consistent with previous results, Alexa647-K3 was not observed within the tumor (FIG. 4d). Approximately 50% of Alexa647-K3-SPG and FITC-SPG observed within the tumor coexist with TRITC-dextran positive cells (FIGS. 4e, f, g), indicating that K3-SPG is phagocytosed in the tumor microenvironment. It is taken up by cells. Some K3-SPGs do not associate with dextran and we speculate that they accumulated passively in the space within the tumor tissue via the vascular permeability and retention (EPR) effect. ing. K3-SPG i. v. To test the importance of phagocytes for treatment, we injected clodronate liposomes intravenously. The present inventors usually used 100 nm clodronate liposomes instead of 200-300 nm liposomes and injected them to deplete phagocytes in the tumor (Pante, N. et al. Molecular biology). of the cell 13,425-434 (2002); Pante, N. et al. Molecular biology of the cell 13,425-434 (2002)), this injection almost resulted in 2 days of F4 / 80 positive cells in the tumor. It was depleted (Figure 5). Tumor-bearing mice are injected with clodronate liposomes on day 5 (2 days before the first K3-SPG treatment) or not, and mice are treated with K3-SPG as in FIG. 2 (AB). Treated. The previous injection of clodronate liposomes significantly offset the suppression of K3-SPG-mediated tumor growth (p <0.05) (FIG. 4h), whereas the injection of clodronate liposomes itself is PBS There was no effect on tumor growth compared to treated mice. These results indicate that K3-SPG targets phagocytic cells in the tumor microenvironment, and the antitumor effect of K3-SPG is largely dependent on phagocytic cell uptake in the tumor microenvironment. Show.
　（実施例３：腫瘍微小環境におけるＩＬ１２およびＩＦＮ−Ｉサイトカインの両方を産生することは、Ｋ３−ＳＰＧ単剤治療に重要である）
　次に、本実施例では、本発明者らは、Ｋ３−ＳＰＧ単剤治療の成功に必要とされる因子について試験した。 (Example 3: Producing both IL12 and IFN-I cytokines in the tumor microenvironment is important for K3-SPG monotherapy)
Next, in this example, the inventors tested for factors required for successful K3-SPG monotherapy.
　ＩＬ−１２およびＩＦＮ−Ｉなどのサイトカインは、Ｋ３−ＳＰＧ（Ｋｏｂｉｙａｍａ，Ｋ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１１１，３０８６−３０９１　（２０１４））を含むＣｐＧ　ＯＤＮ（Ｋｒｉｅｇ，Ａ．Ｍ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｌｏｇｙ　１６１，２４２８−２４３４　（１９９８）；Ｋｌｉｎｍａｎ，Ｄ．Ｍ．，ｅｔ　ａｌ．Ｉｍｍｕｎｉｔｙ　１１，１２３−１２９　（１９９９）；Ｉｓｈｉｉ，Ｋ．Ｊ．，ｅｔ　ａｌ．Ｃｕｒｒｅｎｔ　ｏｐｉｎｉｏｎ　ｉｎ　ｍｏｌｅｃｕｌａｒ　ｔｈｅｒａｐｅｕｔｉｃｓ　６，１６６−１７４　（２００４））の重要な免疫刺激因子であることが示されている。それゆえ、本発明者らは、ＩＬ−１２およびＩＦＮ−Ｉが、Ｋ３−ＳＰＧの治療による腫瘍縮小に必要とされるかどうか試験した。 Cytokines such as IL-12 and IFN-I include C3-SPG (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)). ODN (Krieg, AM, et al. Journal of immunology 161, 428-2434 (1998); Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, KJ. , Et al. Current opinion in molecular therapeutics 6, 166-174 (2004)). Therefore, we tested whether IL-12 and IFN-I are required for tumor reduction by treatment with K3-SPG.
　Ｉｌ１２ｐ４０およびＩＦＮＡＲ２欠損マウスに、ＥＧ７細胞を０日目に皮下に接種し、図２（Ａ~Ｂ）のようにＰＢＳまたはＫ３−ＳＰＧ（１０μｇ）で３回ｉ．ｖ．処置をした。その後腫瘍縮小に対するＫ３−ＳＰＧの影響を観察した。
　（結果） Ll7p40 and IFNAR2-deficient mice were inoculated subcutaneously with EG7 cells on day 0 and i.p. three times with PBS or K3-SPG (10 μg) as in FIG. 2 (AB). v. Treated. Thereafter, the effect of K3-SPG on tumor shrinkage was observed.
(result)
　結果を図６（Ａ~Ｂ）以下に示す。腫瘍縮小に対するＫ３−ＳＰＧの影響は、ＩＬ−１２ｐ４０およびＩＦＮ−Ｉシグナル伝達に部分的に依存していた（図６ａ、ｂ（図６Ａ））。また、本発明者らは、ＩＬ１２ｐ４０およびＩＦＮＡＲ２の二重欠損（ＤＫＯ）マウスを試験し、Ｋ３−ＳＰＧの影響は、ＤＫＯマウスにおいて完全に抑止されることを見出した（図６ｃ（図６Ａ））。ＩＦＮ−βおよびＩＬ−１２ｐ４０もまた、ＩＨＣ染色により腫瘍内で検出された（図７、図８）。これらのデータは、腫瘍内でのＩＬ１２ｐ４０およびＩＦＮ−Ｉサイトカインの両方の分泌は、Ｋ３−ＳＰＧ媒介性腫瘍抑制に重要であることを示した。 Results are shown in FIG. 6 (AB) and below. The effect of K3-SPG on tumor shrinkage was partially dependent on IL-12p40 and IFN-I signaling (FIGS. 6a, b (FIG. 6A)). We also tested IL12p40 and IFNAR2 double deficient (DKO) mice and found that the effects of K3-SPG were completely abrogated in DKO mice (FIG. 6c (FIG. 6A)). . IFN-β and IL-12p40 were also detected in the tumor by IHC staining (FIGS. 7 and 8). These data indicated that secretion of both IL12p40 and IFN-I cytokines within the tumor is important for K3-SPG-mediated tumor suppression.
　本発明者らはまた、Ｔ細胞およびＢ細胞媒介性の適応免疫応答を完全に欠損しているＲａｇ２マウスを試験した。Ｒａｇ２マウスは、Ｋ３−ＳＰＧ処置をしても、全ての腫瘍成長を制御できなかったが（図６ｄ（図６Ａ））、本発明者らは、Ｋ３−ＳＰＧでの３回の処置の間に、ｒａｇ２欠損マウスは、部分的に腫瘍成長を制御することができたことがわかった（図６ｆ（図６Ａ））。この観察を確かめるために、本発明者らは、ｒａｇ２マウスの６回処置群（７、９、１１日目および１４、１６、１８日目）を作製し、ｒａｇ２マウスのこのプロトコルにおいてより明白な腫瘍の制御を見出した（図６ｆ（図６Ａ））。興味深いことに、ＩＬ１２ｐ４０およびＩＦＮＡＲ２　ＤＫＯマウスは、この広範な処置プロトコルでも、Ｋ３−ＳＰＧ単剤治療に対して完全に非応答だった（図６ｅ（図６Ａ））。これらのデータは、Ｋ３−ＳＰＧの治療は、腫瘍内でＩＬ−１２ｐ４０およびＩＦＮ−Ｉの両方を誘発し、その結果、腫瘍に対する自然免疫応答および適応免疫応答の両方を生じさせたことを示した。 We also tested Rag2 mice that are completely deficient in T cell and B cell mediated adaptive immune responses. Although Rag2 mice were unable to control all tumor growth even with K3-SPG treatment (FIG. 6d (FIG. 6A)), we observed that during the three treatments with K3-SPG It was found that rag2-deficient mice were able to partially control tumor growth (FIG. 6f (FIG. 6A)). To confirm this observation, we created 6 treatment groups of rag2 mice ( days 7, 9, 11 and 14, 16, 18), which is more evident in this protocol for rag2 mice. Tumor control was found (FIG. 6f (FIG. 6A)). Interestingly, IL12p40 and IFNAR2 DKO mice were completely unresponsive to K3-SPG monotherapy even with this extensive treatment protocol (FIG. 6e (FIG. 6A)). These data indicated that K3-SPG treatment induced both IL-12p40 and IFN-I within the tumor, resulting in both an innate and adaptive immune response against the tumor. .
　（実施例４：Ｋ３−ＳＰＧ処置は、ＩＬ１２ｐ４０およびＩＦＮ−Ｉの両方に依存する態様で、腫瘍細胞死を誘発する）
　本実施例では、Ｋ３−ＳＰＧ処置は、ＩＬ１２ｐ４０およびＩＦＮ−Ｉの両方に依存する態様で、腫瘍細胞死を誘発することを実証した。 Example 4: K3-SPG treatment induces tumor cell death in a manner dependent on both IL12p40 and IFN-I
In this example, K3-SPG treatment was demonstrated to induce tumor cell death in a manner that is dependent on both IL12p40 and IFN-I.
　ｒａｇ２マウスにおいて観察される適応免疫によらない部分的な腫瘍成長の抑制、およびＩＬ１２ｐ４０およびＩＦＮＡＲ２　ＤＫＯマウスにおけるその完全な抑止が観察されたため、本発明者らは、広範囲なＫ３−ＳＰＧ処置中の腫瘍−宿主相互作用を試験した。 Due to the observed partial suppression of tumor growth independent of adaptive immunity observed in rag2 mice and its complete suppression in IL12p40 and IFNAR2 DKO mice, we have observed extensive tumors during K3-SPG treatment. -Host interaction was tested.
　本発明者らは、１２日目に取り除かれた脾臓（Ｋ３−ＳＰＧでの３回の処置の次の日）が、ＰＢＳ処置された脾臓と比較して、多量のＣＤ４５陰性細胞を含んでいたことを見出した（図６ｇ（図６Ｂ））。興味深いことに、これらのＣＤ４５陰性細胞は、ＩＬ１２ｐ４０およびＩＦＮＡＲ２　ＤＫＯマウスにおいて顕著に減少した（図６ｇ、ｈ（図６Ｂ））。本発明者らは、これらのＣＤ４５陰性細胞をソートした。このサイズおよび形態は、それらが腫瘍細胞に由来することを強く示した。ＧＦＰマウスにおけるＥＧ７接種実験により、これらのＣＤ４５陰性細胞もまたＧＦＰ陰性であることをさらに確認し、このことは、これらの細胞が腫瘍細胞由来であったことを示した（図９）。ＥＧ７細胞はＣＤ４５を発現しないので、ＣＤ４５が陰性であることもまた、この仮説を支持している。ＨｏｅｃｈｓｔおよびＰＩ染色は、脾臓におけるほとんどのＣＤ４５陰性細胞は、アポトーシスおよびネクローシスの両方の特徴からなる死細胞であった（図６ｉ（図６Ｂ））。これらのデータは、Ｋ３−ＳＰＧにより標的とされる腫瘍食細胞が、腫瘍微小環境においてＩＬ−１２ｐ４０およびＩＦＮ−Ｉを分泌し、これらのサイトカインが腫瘍細胞死を誘発し、それらを循環に放出して最終的に脾臓に捕捉されたことを示した。 We found that the spleen removed on day 12 (next day after 3 treatments with K3-SPG) contained a greater amount of CD45 negative cells compared to the spleen treated with PBS. (FIG. 6g (FIG. 6B)). Interestingly, these CD45 negative cells were markedly reduced in IL12p40 and IFNAR2 DKO mice (FIG. 6g, h (FIG. 6B)). We sorted these CD45 negative cells. This size and morphology strongly indicated that they were derived from tumor cells. EG7 inoculation experiments in GFP mice further confirmed that these CD45 negative cells were also GFP negative, indicating that these cells were derived from tumor cells (FIG. 9). Since EG7 cells do not express CD45, CD45 negative is also supporting this hypothesis. Hoechst and PI staining revealed that most CD45 negative cells in the spleen were dead cells consisting of both apoptotic and necrotic features (FIG. 6i (FIG. 6B)). These data indicate that tumor phagocytes targeted by K3-SPG secrete IL-12p40 and IFN-I in the tumor microenvironment, and these cytokines induce tumor cell death and release them into the circulation. It was shown that it was finally trapped in the spleen.
　（実施例５：放出された腫瘍死細胞は複数の腫瘍抗原に対する抗腫瘍ＣＴＬを誘発する）
　本実施例では、放出された腫瘍死細胞は複数の腫瘍抗原に対する抗腫瘍ＣＴＬを誘発することを実証した。 (Example 5: Released tumor dead cells induce anti-tumor CTL against multiple tumor antigens)
In this example, it was demonstrated that released tumor dead cells induce anti-tumor CTL against multiple tumor antigens.
　Ｋ３−ＳＰＧ処置されたマウスの脾臓において見つかったこれらのＣＤ４５陰性細胞の免疫原性を試験するために、本発明者らはこれらの細胞をソートし、免疫化として無処置マウスに静脈内注射をした。その後、免疫化されたマウスを、ソートされた細胞投与の７日後にＥＧ７腫瘍細胞を移植した。ＣＤ４５陰性細胞免疫化マウスは、ＥＧ７腫瘍の増殖に対して顕著に防御された（図６ｊ（図６Ｂ））。興味深いことに、コントロールマウスおよび免疫化マウスにおけるＯＶＡ２５７テトラマー陽性細胞（図６ｋ（図６Ｂ）における赤の点）は、腫瘍サイズと相関せず（図６ｋ（図６Ｂ）の棒）、ＣＤ４５陰性細胞による免疫化は、単なるＯＶＡ２５７エピトープよりもむしろ、ＥＧ７腫瘍に対するより有効なさらなる免疫応答を誘発したことを示した（図６ｋ（図６Ｂ））。これらの結果は、Ｋ３−ＳＰＧ単剤治療は、ＩＬ−１２およびＩＦＮ−Ｉの両方に依存する腫瘍細胞死を誘発し、この腫瘍死細胞は、抗腫瘍免疫応答のための効率的な免疫原として機能することを示した。 To test the immunogenicity of these CD45 negative cells found in the spleen of K3-SPG treated mice, we sorted these cells and injected intravenously into untreated mice as an immunization. did. The immunized mice were then transplanted with EG7 tumor cells 7 days after sorted cell administration. CD45 negative cell immunized mice were significantly protected against the growth of EG7 tumors (FIG. 6j (FIG. 6B)). Interestingly, OVA257 tetramer positive cells (red dots in FIG. 6k (FIG. 6B)) in control and immunized mice did not correlate with tumor size (FIG. 6k (FIG. 6B) bars) and were due to CD45 negative cells Immunization showed that it elicited a more effective additional immune response against the EG7 tumor, rather than just the OVA257 epitope (Figure 6k (Figure 6B)). These results indicate that K3-SPG monotherapy induces tumor cell death that is dependent on both IL-12 and IFN-I, which is an efficient immunogen for anti-tumor immune responses. As shown to function as.
　（ＣＤ８Ｔ細胞は、Ｋ３−ＳＰＧ媒介性の腫瘍縮小において重要なエフェクターである）
　Ｒａｇ２マウスの結果は、Ｋ３−ＳＰＧの腫瘍抑制効果もまた、適応免疫応答に依存することを示した。それゆえ、本発明者らは、Ｋ３−ＳＰＧ治療に必要なＣＤ８Ｔ細胞を試験した。ｉｎ　ｖｉｖｏにおけるＣＤ８　Ｔ細胞の枯渇は、Ｋ３−ＳＰＧの抗腫瘍効果を顕著に抑止し（図１０ａ（図１０Ａ））、ＣＤ８Ｔ細胞がこのＫ３−ＳＰＧ治療における重要なエフェクター細胞であることを示している。また、Ｋ３−ＳＰＧによる腫瘍縮小は、Ｂａｔｆ３（交差提示ＣＤ８α＋　ＤＣを欠損している）にも依存し（図１０ｂ（図１０Ａ））、Ｋ３−ＳＰＧ単剤治療が、ＣＤ８α＋　ＤＣ媒介性交差提示をも増強したことを示している。本発明者らは、ＣＤ８Ｔ細胞の腫瘍浸潤と腫瘍成長との間の明らかな関連性を観察した。ＣＤ８Ｔ細胞は、Ｋ３−ＳＰＧ　ｉ．ｖ．群において腫瘍領域で蓄積したが、ｉ．ｄ．群では蓄積しなかった（図１０ｃ（図１０Ａ））。 (CD8 T cells are important effectors in K3-SPG-mediated tumor shrinkage)
The results of Rag2 mice showed that the tumor suppressive effect of K3-SPG is also dependent on the adaptive immune response. Therefore, we tested CD8 T cells required for K3-SPG treatment. In vivo CD8 T cell depletion markedly abrogated the antitumor effect of K3-SPG (FIG. 10a (FIG. 10A)), indicating that CD8 T cells are important effector cells in this K3-SPG treatment Yes. Tumor reduction by K3-SPG also depends on Batf3 (deficient in cross-presented CD8α + DC) (FIG. 10b (FIG. 10A)), and K3-SPG monotherapy is CD8α + DC-mediated crossover. It also shows that presentation has been enhanced. The inventors observed a clear link between tumor invasion of CD8 T cells and tumor growth. CD8 T cells are expressed as K3-SPG i. v. Accumulated in the tumor area in the group i. d. There was no accumulation in the group (FIG. 10c (FIG. 10A)).
　最後に、本発明者らは、これらのＣＤ８Ｔ細胞が腫瘍領域に進入するために必要なものを試験した。ＷＴマウスおよびＩｌ１２ｐ４０−Ｉｆｎａｒ２　ＤＫＯマウスに、ＥＧ７細胞を０日目に接種し、７、９、および１１日目にＫ３−ＳＰＧまたはＰＢＳでｉ．ｖ．処置した。１４日目に、ＣＤ８α＋Ｔ細胞を、これらのマウスの脾臓から精製し、Ｘｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で染色し、Ｋ３−ＳＰＧ処置された（７、９、および１１日目）別のＥＧ７保有マウスに移し（接種１４日後）、その後Ｘｅｎｏｌｉｇｈｔ　ＤｉＲ（登録商標）で標識されたＣＤ８Ｔ細胞の分布をＩＶＩＳで１５日目に分析した（図１１）。１５日目に、未処置腫瘍を保有するドナーマウス由来のＣＤ８Ｔ細胞は、Ｋ３−ＳＰＧで処置されてもＷＴレシピエントマウスの腫瘍部位で蓄積しなかった（図１０ｄ（図１０Ｂ）、ＩＩ）。他方で、Ｋ３−ＳＰＧで処置された腫瘍を保有するドナーマウス由来のＣＤ８Ｔ細胞は、レシピエントマウスの腫瘍部位で検出され（図１０ｄ（図１０Ｂ）、Ｉ）、Ｋ３−ＳＰＧ単剤治療は、腫瘍微小環境へと移動して浸潤し得る抗腫瘍ＣＤ８Ｔ細胞を誘発したことを示した。これらのｉｎ　ｖｉｖｏ活性化ＣＤ８Ｔ細胞は、ＤＫＯレシピエントマウスの腫瘍微小環境に進入することが可能であった（図１０ｅ（図１０Ｂ））。たとえ、ＩＬ−１２およびＩＦＮ−Ｉは、全身性のＫ３−ＳＰＧ単剤治療による自然免疫およびＣＤ８Ｔ細胞の誘発に重要であっても、この結果は、ＣＤ８Ｔ細胞がＫ３−ＳＰＧ治療で活性化されると、腫瘍微小環境におけるＩＬ−１２およびＩＦＮ−Ｉサイトカインの分泌がＣＤ８Ｔ細胞の腫瘍浸潤に必ずしも必要であるとは限らないことを示した。まとめると、これらの結果は、腫瘍特異的ＣＤ８Ｔ細胞の活性化が、腫瘍への浸潤に十分であることを示した。驚くべきことに、これらのＣＤ８Ｔ細胞の浸潤は、腫瘍微小環境におけるサイトカインの産生に依存しない。
　（考察） Finally, we tested what is necessary for these CD8 T cells to enter the tumor area. WT and Il12p40-Ifnar2 DKO mice are inoculated with EG7 cells on day 0 and i.p. with K3-SPG or PBS on days 7, 9, and 11. v. Treated. On day 14, CD8α + T cells were purified from the spleens of these mice, stained with Xenlight DiR®, and K3-SPG treated (day 7, 9, and 11) another EG7 possessed The distribution of CD8 T cells labeled with Xenolight DiR® was analyzed by IVIS on day 15 (FIG. 11). On day 15, CD8 T cells from donor mice bearing untreated tumors did not accumulate at the tumor site of WT recipient mice even when treated with K3-SPG (FIG. 10d (FIG. 10B), II). On the other hand, CD8 T cells from donor mice bearing tumors treated with K3-SPG were detected at the tumor site of recipient mice (FIG. 10d (FIG. 10B), I), and K3-SPG monotherapy was We showed that we induced anti-tumor CD8 T cells that could migrate to and invade the tumor microenvironment. These in vivo activated CD8 T cells were able to enter the tumor microenvironment of DKO recipient mice (FIG. 10e (FIG. 10B)). Even though IL-12 and IFN-I are important for innate immunity and CD8 T cell induction by systemic K3-SPG monotherapy, this result indicates that CD8 T cells are activated by K3-SPG treatment Thus, it was shown that secretion of IL-12 and IFN-I cytokines in the tumor microenvironment is not necessarily required for tumor invasion of CD8 T cells. Taken together, these results indicated that tumor-specific CD8 T cell activation was sufficient for tumor invasion. Surprisingly, the infiltration of these CD8 T cells is independent of cytokine production in the tumor microenvironment.
(Discussion)
　本発明者らは、新規ながん免疫療法の可能性を示した。これはＣｐＧが腫瘍微小環境における食細胞に標的化されるという新しい治療である（図１２）。ＴＬＲ９の刺激を介して、ＣｐＧが免疫細胞による免疫応答を誘発し、特にマクロファージおよびＤＣを活性化する（Ｋｌｉｎｍａｎ，Ｄ．Ｍ．，ｅｔ　ａｌ．Ｉｍｍｕｎｉｔｙ　１１，１２３−１２９　（１９９９）；Ｉｓｈｉｉ，Ｋ．Ｊ．，ｅｔ　ａｌ．Ｃｕｒｒｅｎｔ　ｏｐｉｎｉｏｎ　ｉｎ　ｍｏｌｅｃｕｌａｒ　ｔｈｅｒａｐｅｕｔｉｃｓ　６，１６６−１７４（２００４））。この活性化は、抗がん免疫応答に非常に重要である。以前の報告では、ＣｐＧを直接腫瘍内に投与しなければならなかったが、ＳＰＧとＣｐＧの複合体ではＤＤＳ機能が追加され全身投与でも腫瘍内投与と同等もしくはそれ以上の有効性が示され（Ｓｃｈｅｔｔｉｎｉ，Ｊ．，ｅｔ　ａｌ．Ｃａｎｃｅｒ　ｉｍｍｕｎｏｌｏｇｙ，ｉｍｍｕｎｏｔｈｅｒａｐｙ　：　ＣＩＩ　６１，２０５５−２０６５　（２０１２）；Ｌｏｕ，Ｙ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｔｈｅｒａｐｙ　（Ｈａｇｅｒｓｔｏｗｎ，Ｍｄ．：　１９９７）３４，２７９−２８８　（２０１１）；Ｎｉｅｒｋｅｎｓ，Ｓ．，ｅｔ　ａｌ．ＰＬｏＳ　Ｏｎｅ　４，ｅ８３６８　（２００９）；Ｈｅｃｋｅｌｓｍｉｌｌｅｒ，Ｋ．，ｅｔ　ａｌ．Ｊｏｕｒｎａｌ　ｏｆ　ｉｍｍｕｎｏｌｏｇｙ　１６９，３８９２−３８９９　（２００２）；Ｉｓｈｉｉ，Ｋ．Ｊ．，ｅｔ　ａｌ．Ｃｕｒｒｅｎｔ　ｏｐｉｎｉｏｎ　ｉｎ　ｍｏｌｅｃｕｌａｒ　ｔｈｅｒａｐｅｕｔｉｃｓ　６，１６６−１７４　（２００４））、本発明者らはこの課題を解決した。ナノ粒子の形成によるＳＰＧとＣｐＧとの複合体は（Ｋｏｂｉｙａｍａ，Ｋ．，ｅｔ　ａｌ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１１１，３０８６−３０９１　（２０１４））、ｉｎ　ｖｉｖｏにおいて安定化することが可能であった。この効果が腫瘍環境を標的とすることを可能とすることがわかり、ＴＬＲ９免疫担当細胞が腫瘍環境に供された。本発明者らによって開発されたこの新規ＣｐＧは、ナノ粒子を形成させるために食細胞によって貪食される。 The present inventors have shown the possibility of a novel cancer immunotherapy. This is a new treatment where CpG is targeted to phagocytes in the tumor microenvironment (FIG. 12). Through stimulation of TLR9, CpG induces an immune response by immune cells, and in particular activates macrophages and DCs (Klinman, DM, et al. Immunity 11, 123-129 (1999); Ishii, K J., et al. Current opinion in molecular therapeutics 6, 166-174 (2004)). This activation is very important for the anti-cancer immune response. In previous reports, CpG had to be administered directly into the tumor, but the DPG function was added to the SPG and CpG complex, and systemic administration is as effective or better than intratumoral administration ( Schettini, J., et al. Cancer immunology, immunotherapy: CII 61, 2055-2065 (2012); Lou, Y., et al. Journal of immunotherapy (Hagerstown, 88, 19: 2) 19: 2 (19): 19: 2 Nierkens, S., et al. PLoS One 4, e8368 (2009); Heckelsmiller, K., et al. Journal of immunology 169, 892-3899 (2002); Ishii, K.J., et al.Current opinion in molecular therapeutics 6,166-174 (2004)), the present inventors have solved this problem. The complex of SPG and CpG due to the formation of nanoparticles (Kobiyama, K., et al. Proc. Natl. Acad. Sci. USA 111, 3086-3091 (2014)) is stable in vivo. It was possible to It was found that this effect made it possible to target the tumor environment, and TLR9 immunocompetent cells were subjected to the tumor environment. This novel CpG developed by the inventors is phagocytosed by phagocytic cells to form nanoparticles.
　その後、腫瘍環境において、この新規ＣｐＧを貪食した食細胞は、ＩＦＮおよびＩＬ−１２などのサイトカインを産生する。これらのサイトカインが腫瘍環境において誘発されることは非常に重要である。以前の報告には、腫瘍環境を直接標的としたＩＦＮβ治療は、樹状細胞を腫瘍内に移動させ、腫瘍内微小環境内の抗原交差提示を増加させることによりＣＴＬを再活性化させることが記載されている。これらのサイトカインは、腫瘍細胞の細胞死を引き起こす。さらに、本発明者らは、この効果が自然免疫の活性化によって及ぼされることを見出した。この細胞死は、非常に重要な役割を担っている。これは自然免疫と適応免疫との間の連携をなしていた。腫瘍細胞死が腫瘍微小環境から放出されることによって、獲得免疫を誘発する。この免疫原性腫瘍細胞死は、複数の細胞障害性Ｔリンパ球を誘発する。上記のようにｉｎ　ｖｉｖｏにおける腫瘍特異的に誘発されるＣＴＬは、腫瘍に応答して腫瘍微小環境に浸潤し得る。この抗腫瘍免疫システムは内因性抗原を使用し、がん免疫療法の障壁である免疫編集に対処し得ると考えられる。 Thereafter, phagocytes phagocytosed with this novel CpG in the tumor environment produce cytokines such as IFN and IL-12. It is very important that these cytokines are induced in the tumor environment. Previous reports described that IFNβ treatment directly targeting the tumor environment reactivates CTL by moving dendritic cells into the tumor and increasing antigen cross-presentation within the tumor microenvironment. Has been. These cytokines cause tumor cell death. Furthermore, the present inventors have found that this effect is exerted by activation of innate immunity. This cell death plays a very important role. This was a link between innate and adaptive immunity. Tumor cell death is released from the tumor microenvironment, thereby inducing acquired immunity. This immunogenic tumor cell death induces multiple cytotoxic T lymphocytes. As described above, tumor-specifically induced CTL in vivo can invade the tumor microenvironment in response to the tumor. This anti-tumor immune system may use endogenous antigens to address immune editing, a barrier to cancer immunotherapy.
　Ｋ３−ＳＰＧ単剤治療後の腫瘍細胞の循環は、腫瘍に対するこの処置効果の優れたバイオマーカーとして機能し得る。 The circulation of tumor cells after K3-SPG single agent treatment can function as a biomarker with excellent treatment effect on tumors.
　（実施例６：製剤例）
　製剤化する場合の組成をご教示ください
　製剤は、例えば、７．２２ｍｇのＫ３−ｄＡ４０（配列番号２）を水（３．７ｍＬ）に溶解し、ＳＰＧ（１５ｍｇ）を０．２５Ｎ　ＮａＯＨ（１ｍＬ）に溶解した。１ｍＬの容積の３３０ｍＭ　ＮａＨ２ＰＯ４をＤＮＡ溶液に加え、次いで、ＳＰＧ溶液をこのＤＮＡ／ＮａＨ２ＰＯ４溶液に加え、４℃で一晩維持して、複合体化を完了させた。モル比（ＭＳＰＧ／ＭＤＮＡ）は０．２７に固定して製造することができる。 (Example 6: Formulation example)
Please tell me the composition when formulating. For example, 7.22 mg of K3-dA 40 (SEQ ID NO: 2) is dissolved in water (3.7 mL), and SPG (15 mg) is dissolved in 0.25N NaOH (1 mL). ). A 1 mL volume of 330 mM NaH 2 PO 4 was added to the DNA solution and then the SPG solution was added to the DNA / NaH 2 PO 4 solution and maintained at 4 ° C. overnight to complete the complexation. The molar ratio (M SPG / M DNA ) can be prepared by fixing at 0.27.
　製剤に使用した薬剤は、ジーンデザイン、ｉｎｖｉｖｏｇｅｎ、Ｗａｋｏなどから入手することができる。 Drugs used in the formulation can be obtained from Gene Design, Invivogen, Wako and others.
　以上のように、本発明の好ましい実施形態を用いて本発明を例示してきたが、本発明は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。 As described above, the present invention has been exemplified by using the preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the scope of the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.
　本発明により、単剤として使用できる新たな形態の抗がん剤が提供される。したがって、本発明の複合体は、抗がん剤として医薬分野において有用である。 The present invention provides a new form of anticancer agent that can be used as a single agent. Therefore, the complex of the present invention is useful in the pharmaceutical field as an anticancer agent.
配列番号１：Ｋ３
配列番号２：Ｋ３−ｄＡ４０
配列番号３：ｄＡ４０−Ｋ３
配列番号４：Ｋ３−ｄＡ２０
配列番号５：Ｋ３−ｄＡ２５
配列番号６：Ｋ３−ｄＡ３０
配列番号７：Ｋ３−ｄＡ３５
SEQ ID NO: 1 K3
SEQ ID NO: 2: K3-dA 40
SEQ ID NO: 3: dA 40 -K3
SEQ ID NO: 4: K3-dA20
SEQ ID NO: 5: K3-dA25
SEQ ID NO: 6: K3-dA30
SEQ ID NO: 7: K3-dA35

Claims (23)

  1. （ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
    （ｂ）β—１，３−グルカンとを
    含む、複合体を含む抗がん剤。     (A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
    (B) An anticancer agent containing a complex containing β-1,3-glucan.
  2. 前記抗がん剤は、がん抗原なしで投与されることを特徴とする、請求項１に記載の抗がん剤。 The anticancer agent according to claim 1, wherein the anticancer agent is administered without a cancer antigen.
  3. 前記抗がん剤は、細網内皮系および／またはリンパ節に送達されるように投与されることを特徴とする、請求項１または２に記載の抗がん剤。 The anticancer agent according to claim 1 or 2, wherein the anticancer agent is administered so as to be delivered to a reticuloendothelial system and / or a lymph node.
  4. 前記細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む、請求項３に記載の抗がん剤。 The anticancer agent according to claim 3, wherein the reticuloendothelial system and / or lymph node includes tumor and phagocytic cells.
  5. 前記細網内皮系は脾臓および／または肝臓を含む、請求項３または４に記載の抗がん剤。 The anticancer agent according to claim 3 or 4, wherein the reticuloendothelial system includes spleen and / or liver.
  6. 前記抗がん剤は、がん抗原なしで投与されることを特徴とする、請求項１~５のいずれか１項に記載の抗がん剤。 The anticancer agent according to any one of claims 1 to 5, wherein the anticancer agent is administered without a cancer antigen.
  7. 前記投与は全身性投与を含む、請求項２~６のいずれか１項に記載の抗がん剤。 The anticancer agent according to any one of claims 2 to 6, wherein the administration comprises systemic administration.
  8. 前記全身性投与は、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与、および腫瘍内投与から選択される、請求項７に記載の抗がん剤。 The anticancer agent according to claim 7, wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
  9. 前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）からなる群より選択される、請求項１~８のいずれか１項に記載の抗がん剤。 The oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), The anticancer agent according to any one of claims 1 to 8, which is selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  10. 前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、請求項１~９のいずれか１項に記載の抗癌剤。 The anticancer agent according to any one of claims 1 to 9, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran, and laminaran. .
  11. 前記複合体は、Ｋ３−ＳＰＧである、請求項１~１０のいずれか１項に記載の抗がん剤。 The anticancer agent according to any one of claims 1 to 10, wherein the complex is K3-SPG.
  12. （ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
    （ｂ）β—１，３−グルカンとを
    含む、複合体
    を含む、がんの死細胞を細網内皮系および／またはリンパ節に集積させるための組成物。     (A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
    (B) A composition for accumulating dead cells of cancer in the reticuloendothelial system and / or lymph nodes, comprising a complex containing β-1,3-glucan.
  13. 前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）からなる群より選択される、請求項１２に記載の組成物。 The oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), 13. The composition of claim 12, wherein the composition is selected from the group consisting of K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  14. 前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、請求項１２または１３に記載の組成物。 The composition according to claim 12 or 13, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran.
  15. 前記複合体は、Ｋ３−ＳＰＧである、請求項１２~１４のいずれか１項に記載の組成物。 The composition according to any one of claims 12 to 14, wherein the complex is K3-SPG.
  16. 前記細網内皮系および／またはリンパ節は、腫瘍および貪食細胞を含む、請求項１２~１５のいずれか１項に記載の組成物。 The composition according to any one of claims 12 to 15, wherein the reticuloendothelial system and / or lymph node contains a tumor and a phagocytic cell.
  17. 前記細網内皮系は脾臓および／または肝臓を含む、請求項１２~１６のいずれか１項に記載の組成物。 The composition according to any one of claims 12 to 16, wherein the reticuloendothelial system includes spleen and / or liver.
  18. 前記投与は全身性投与を含む、請求項１２~１７のいずれか１項に記載の組成物。 The composition according to any one of claims 12 to 17, wherein the administration comprises systemic administration.
  19. 前記全身性投与は、静脈内投与、腹腔内投与、経口投与、皮下投与、筋肉内投与、および腫瘍内投与から選択される、請求項１８に記載の組成物。 19. The composition of claim 18, wherein the systemic administration is selected from intravenous administration, intraperitoneal administration, oral administration, subcutaneous administration, intramuscular administration, and intratumoral administration.
  20. （ａ）ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチド及びポリデオキシアデニル酸を含む、オリゴデオキシヌクレオチドであって、ポリデオキシアデニル酸が、ヒト化Ｋ型ＣｐＧオリゴデオキシヌクレオチドの３’側に配置されている、オリゴデオキシヌクレオチドと、
    （ｂ）β—１，３−グルカンとを
    含む、インターロイキン１２（ＩＬ１２）および／またはインターフェロン（ＩＦＮ）γの発現またはその促進のための組成物。     (A) an oligodeoxynucleotide comprising a humanized K-type CpG oligodeoxynucleotide and polydeoxyadenylic acid, wherein the polydeoxyadenylic acid is located 3 ′ of the humanized K-type CpG oligodeoxynucleotide; Oligodeoxynucleotides;
    (B) A composition for expression or promotion of interleukin 12 (IL12) and / or interferon (IFN) γ, comprising β-1,3-glucan.
  21. 前記オリゴデオキシヌクレオチドはＫ３（配列番号１）、Ｋ３−ｄＡ４０（配列番号２）、ｄＡ４０−Ｋ３（配列番号３）、Ｋ３−ｄＡ２０（配列番号４）、Ｋ３−ｄＡ２５（配列番号５）、Ｋ３−ｄＡ３０（配列番号６）およびＫ３−ｄＡ３５（配列番号７）である、請求項２０に記載の組成物。 The oligodeoxynucleotide is K3 (SEQ ID NO: 1), K3-dA 40 (SEQ ID NO: 2), dA 40 -K3 (SEQ ID NO: 3), K3-dA20 (SEQ ID NO: 4), K3-dA25 (SEQ ID NO: 5), 21. The composition of claim 20, which is K3-dA30 (SEQ ID NO: 6) and K3-dA35 (SEQ ID NO: 7).
  22. 前記β—１，３−グルカンはシゾフィラン（ＳＰＧ）、レンチナン、スクレログルカン、カードラン、パーキマン、グリホランおよびラミナランからなる群より選択される、請求項２０または２１に記載の組成物。 The composition according to claim 20 or 21, wherein the β-1,3-glucan is selected from the group consisting of schizophyllan (SPG), lentinan, scleroglucan, curdlan, parkan, glyphoran and laminaran.
  23. 前記複合体は、Ｋ３−ＳＰＧである、請求項２０~２２のいずれか１項に記載の組成物。 The composition according to any one of claims 20 to 22, wherein the complex is K3-SPG.
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